Methods for inducing an immune response by administering activated mesenchymal stem cells

ABSTRACT

The present invention provides methods or kits with inflammatory cytokines to pretreat 1-ISCs to augment their immune modulatory effect, in prevention and treatment of various diseases such as multiple sclerosis, arthritis, lupus, sepsis, hepatitis, cirrhosis, Parkinson&#39;s disease, chronic infections, and GvHD. The present invention relates to novel methods for enhancing the immunosuppressive or the immune stimulatory activities of mesenchymal stem cells (JvfSCs).

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a divisional of U.S. patent application Ser.No. 16/100,545, filed Aug. 10, 2018, which is a divisional of U.S.patent application Ser. No. 14/652,324 filed Jun. 15, 2015, now U.S.Pat. No. 10,046,011 issued Aug. 14, 2018, which is the U.S. NationalPhase of International Patent Application Serial No. PCT/US13/75208,filed Dec. 14, 2013, which claims the benefit of priority under 35U.S.C. § 119(e) of U.S. Provisional Application Ser. No. 61/737,616,filed Dec. 14, 2012. International Patent Application Serial No.PCT/US13/75208 is also a continuation-in-part of U.S. application Ser.No. 12/362,847, filed Jan. 30, 2009, now U.S. Pat. No. 8,685,728 issuedApr. 1, 2014, which claims the benefit of priority under 35 U.S.C. §119(e) of U.S. Provisional Application Ser. No. 61/063,288, filed Jan.31, 2008. The entire disclosures of the applications noted above areincorporated herein by reference.

GOVERNMENT INTERESTS

This invention was made with government support under grant numberGM866889, DE014913, and DE019932 from the National Institutes of Health,and stem cell grants from New Jersey Commission on Science andTechnology (NJCST-2042-014-84)

CROSS-REFERENCE TO A SEQUENCE LISTING

This application includes a “Sequence Listing” which is provided as anelectronic document having the file name “096738.00688_ST25.txt” (3416bytes, created Jan. 14, 2021), which is herein incorporated by referencein its entirety.

FIELD OF THE INVENTION

The present invention relates to novel methods for enhancing theimmunosuppressive or the immune stimulatory activities of mesenchymalstem cells (MSCs).

BACKGROUND OF THE INVENTION

Cellular therapy involves administration of living cells for any purposeincluding diagnostic or preventive purposes and of any condition, forexample regenerative medicine, transplantation and even cancer. Stemcells are believed to have tremendous potential in cell therapy.However, its effective use in a clinical setting has been elusive forvariety of reasons.

Stem cells have two distinct characteristics that distinguish them fromother cell types. First, they are unspecialized and can self-renew forlong periods without significant changes in their general properties.Second, under certain physiologic or experimental conditions, stem cellscan be induced to differentiate into various specialized cell types.Thus, stem cells hold a great promise for regenerative medicine. Thereare two major types of stem cells: embryonic stem (ES) cells and adultstem cells.

Adult stem cells exist in many mature tissues, such as bone marrow,muscle, fat and brain. While most studies of adult stem cells havefocused on CD34+ hematopoietic stem cells, the distinct lineage of CD34−fibroblast-like mesenchymal stem cells (MSCs), especially those derivedfrom bone marrow, have attracted significant attention from basic andclinical investigators (Chen, et al. (2006) Immunol. Cell Biol.84:413-421; Keating (2006) Curr. Opin. Hematol. 13:419-425; Pommey &Galipeau (2006) Bull. Cancer 93:901-907). Bone marrow-derived MSCs havebeen shown to differentiate into several different cell types of tissue,such as cartilage, bone, muscle, and adipose tissue (Barry & Murphy(2004) Int. J. Biochem. Cell Biol. 36:568-584; Le Blanc & Ringden (2006)Lancet 363:1439-1441).

Mesenchymal stem cells have great potential for regenerative medicineand autoimmune disorders, and have been evaluated in clinical trials totreat many different kinds of diseases, including liver fibrosis,diabetes, GvHD, and Crohn's disease. MSCs can help successfulengraftment of transplanted bone marrow and cells differentiated fromembryonic stem cells or induced pluripotent stem (iPS) cells.Accordingly, the immune suppressive behavior of MSCs can provide abeneficial method in combating such conditions.

From another angle, the immune system plays a key role in combatingtumor development and progression. Tumors are always accompanied by animmunosuppressive microenvironment. MSCs have an intrinsic ability tospecifically migrate into tumors, and have been suggested as atumor-specific vector to deliver anti-tumor agents. In fact, MSCs havebeen genetically engineered to express various anti-tumor factors,including type I interferon, TRAIL, IL-12, and LIGHT, and have beenshown to possess potent anti-tumor effect in animal models. Thus,enhancing anti-tumor immune responses by using the MSC guidedstimulatory affects effects holds great promise for further cancertherapy.

The underlying in vivo mechanisms through which MSCs modulates immuneresponse, suppression or inducement are largely unknown. Moreimportantly, the clinical effects of MSCs vary significantly dependingon the physiological and pathological status of the host and themicroenvironment experienced by MSCs themselves. Thus, there exists aneed to further understand and develop regimens to successfully employthe immune modulatory effects of MSCs in clinical settings.

SUMMARY OF THE INVENTION

The present invention describes methods for suppressing and inducingimmune response by trained populations of MSC. The present inventionalso provides a new source of immune adjuvants using gene modified MSCs.

For use therapeutically, the pharmaceutical composition of the inventioncan be provided as a kit. A kit of the invention contains apharmaceutically acceptable carrier; an isolated population ofmesenchymal stem cells; isolated IFN gamma (IFNγ); isolated IL-1 alpha(IL-1 α); Type 1 interferons (IFN-I such as IFN-α (alpha), IFN-β(beta)), Transforming growth factor beta (TGF β), Fibroblast growthfactor (FGF), isolated interleukin-17 A (IL17-A) and Tumor necrosisfactor (TNF). In another aspect, the kit can contain instructions forusing the kit in a method for attenuating an immune response and/orinducing or boosting an immune response. In yet another embodiment, thekit contains a pharmaceutically acceptable carrier, inhibitors ofimmunosuppressive molecules (such as NO synthases(iNOS)/indoleamine-2,3-dioxygenase (IDO) inhibitors), other cytokines ortherapeutic formulations for boosting or suppressing an immune response.

In one aspect of the invention, composition containing isolated purifiedMSCs, IFNγ and IL-17A are described in admixture with a pharmaceuticallyacceptable carrier. The present invention also provides a compositioncomprising isolated MSCs, IFNγ, TNF α, IL-1 and IL-17 in admixture witha pharmaceutically acceptable carrier.

In another aspect of the present invention, methods for modulating animmune response are described by administering an effective amount of acomposition containing isolated MSCs that have been treated with IFNγand any one of the cytokines IL-1 α; an IFN-I, TGF β, FGF, TNF α orIL-17 and any combinations thereof to a subject in need of a treatmentfor suppressing or inducing the subject's immune response. In anotherembodiment, methods of enhancing immunosuppression in a subject byadministering an effective amount of a composition containing isolatedMSC that have been treated with IFNγ and any one of the cytokines IL-1α, β; TNF α or IL-17 as compared to a subject that has not received suchtreatment or receives anti-inflammatory drugs including corticosteroidsor non-steroidal anti inflammatory drugs to suppress immunity.

The preferred methods and materials are described below in exampleswhich are meant to illustrate, not limit, the invention. Skilledartisans will recognize methods and materials that are similar orequivalent to those described herein, and that can be used in thepractice or testing of the present invention. Other features andadvantages of the invention will be apparent from the detaileddescription, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 . is a graph showing that immunosuppression by MSCs is induced byproinflammatory cytokines. Cloned MSCs were supplemented with theindicated combinations of recombinant cytokines (20 ng/ml each) for 8hours, then co-cultured with CD4+ T cell blasts at a 1:20 ratio (MSC:Tcells), and proliferation assessed after an additional 8 hours. Valuesrepresent means±SD of five wells from a representative of threeexperiments with different clones. *p<0.001.

FIG. 2 . is a graph showing that iNOS-Deficient MSCs Boost DTH. C57BL/6mice were immunized with OVA in complete Freund's adjuvant by tail baseinjection. Mice were challenged in the footpad with 200 g aggregated OVAadministered with or without wild-type or iNOS^(−/−) MSCs (2.5×10⁵cells) on day 7. Footpad thickness increment was determined after 24hours as a measure of DTH. Data shown are means±SD of a representativeof three experiments. *p<0.005 vs. OVA alone.

FIG. 3 . is a graph showing that MSCs Prevent GvHD in a Manner Dependenton Inflammatory Cytokines and NO. Recipient mice (C57BL/6×C3H, F1) werelethally irradiated and injected i.v. with C57BL/6 bone marrow cellsplus spleenocytes. On days 3 and 7 after bone marrow transplantation,recipients were administered with the indicated MSCs. For some wild-typeMSC groups, L-NMMA, anti-IFNγ or a 3-antibody cocktail against TNFα,IL-1α, and IL-1β, (3 Abs) were injected i.p. Survival was monitoreddaily for 12 weeks.

FIG. 4 . is a graph showing that lymphoma stromal cells (LSCs) promotelymphoma development in a NO-dependent manner. 355 B-cell lymphoma cellline (C3H-gld/gld background, 0.5×10⁶ cells/mouse) was co-injected withgld/gld mice-derived lymphoma stromal cells (C3H background, P5,0.25×10⁶ cells/mouse) by tail-vein i.v. on day 0.1400 W (NOS inhibitor,0.1 mg/mouse) was injected on day 0, 2, 4, 8, 12, 16, 20, 24, and 28 byi.p. Mice survival was recorded when mice were moribund.

FIG. 5 . is a graph showing that the combination of NOS inhibitor withIFNγ promotes mouse melanoma therapy. B16-F0 melanoma cells wereinjected into C57BL/6 mice on day 0 by i.v. (0.5×10⁶ cells/mouse). IFNγ(250 ng/mouse) and 1400 W (NOS inhibitor, 0.1 mg/mouse) wereadministrated by i.p. injection on day 4, 8, 12, 16, 20. Mice survivalwas recorded when mice were moribund.

FIG. 6 . IL-17A greatly enhances inflammatory cytokine-induced iNOSexpression in mouse BM-MSCs at both mRNA and protein levels. BM-MSCswere treated with the indicated cytokines. The iNOS gene expression wasmeasured by real-time PCR.

FIG. 7 . IL-17A significantly promoted MSC-mediated immunosuppressiveeffect. MSC cells and T-cell hybridoma A1.1 cell line were co-culturedat a ratio of 1:20. The co-cultures were supplemented with IFNγ+ TNF α,or IL-17A+IFNγ+TNFα. Cell proliferation was measured by the cell densityindicated by O.D. 570 nm.

FIGS. 8A and 8B. IL-17A prevented the decay of iNOS mRNA, (FIG. 8A) theiNOS mRNA stability was measured at different time points afteractinomycin D treatment in IFNγ+TNFα and IFNγ+TNFα+IL-17A treatmentgroups. (FIG. 8B) iNOS expression enhancing effect of IL-17A atdifferent times after IFNγ and TNFα treatment.

FIGS. 9A, 9B, 9C, 9D, 9E, 9F and 9G. (FIG. 9A). Cloned MSCs were firsttreated with the indicated combinations of recombinant cytokines IFNγ,TNFα, IL-17A (2 ng/ml each) for 12 hr, then cocultured with CD4⁺ T cellblasts at a 1:20 ratio (MSC: T cells), and proliferation was assessed by³H-thymidine incorporation after an additional 12 hr. (FIG. 9B). ThemRNA expression of IL-17 receptor family members in MSCs or Raw 264.7(Macrophages) were examined by RT-PCR. NC: No RT. (FIG. 9C). Surfaceexpression of IL-17RA was detected by immunofluoresence or flowcytometry in cloned MSCs. (FIGS. 9D and 9E). MSCs were first treatedwith IFNγ and TNFα with or without IL-17A (10 ng/ml), IFNγ and TNFα weresupplemented at different cytokine concentrations, for 12 hr, and thencocultured with CD4⁺ T cell blasts (FIG. 9D) or T cell hybridoma A1.1cells (FIG. 9E) at a ratio of 1:20 for 12 hr. T cell proliferation wasmeasured by ³H-Tdr incorporation. (FIG. 9F). MSCs were first treatedwith IFNγ and TNFα (2 ng/ml) with graded concentrations of IL-17A, for12 hr, and then cocultured with T cell hybridoma A1.1 cells at a ratioof 1:10 for 12 hr. T cell proliferation was measured by ³H-thymidineincorporation. (FIG. 9G). MSCs were cocultured with fresh C57BL/6splenocytes plus anti-CD3, anti-CD28, and antibodies against IL-17A, ata 1:20 or 1:40 ratio (MSCs: splenocytes), for 48 hr, and then cellproliferation was assessed by ³H-thymidine incorporation. Proliferationvalues represent means±SEM of three wells from a representative of threeexperiments.

FIGS. 10A, 10B, 10C, 10D and 10E. (FIGS. 10A and 10C). MSCs werecultured with different combinations of inflammatory cytokines IFNγ,TNFα, IL-17A (10 ng/ml) for 12 hr, and then cells were harvested for RNAextraction. The mRNA expression levels of inflammatory molecules iNOS,IL-6, CXCL1 (FIG. 10A) and chemokines CCL2, CCL5, CXCL9, CXCL10 (FIG.10C) were detected by quantitative RT-PCR. (FIG. 10B). MSCs werecultured with different combinations of inflammatory cytokines IFNγ,TNFα, IL-17A (10 ng/ml) for 24 hr, and the protein level of iNOS wasdetected by Western Blot. (FIG. 10D). MSCs were supplemented withSup-CD3 or Sup-CD3 pretreated with antibodies against IL-17A, and cellswere collected for RNA extraction after 12 hr. The expression of iNOS,IL-6, CXCL1, CCL2, CCL5, CXCL9 and CXCL10 were measured by quantitativeRT-PCR. Sup-CD3: supernatant from splenocytes activated by anti-CD3 andanti-CD28. (FIG. 10E). MSCs were treated with Sup-CD3 or Sup-CD3pretreated with antibodies against IL-17A, and the protein level of iNOSwas assessed by Western Blot after 24 hr. mRNA expression values aremeans±SEM of three wells from a representative of three independentexperiments. Western Blot data is from a representative of threeindependent experiments.

FIGS. 11A, 11B, 11C and 11D. (FIG. 11A) MSCs with Act1 knockdown orcontrol were treated with IL-17A for different time and thephosphorylation levels of IκBα, ERK, p65, JNK were assessed by WesternBlot. (FIG. 11B) MSCs with Act1 knockdown or control were treated withIFNγ and TNFα with or without IL-17A (all cytokines supplemented at 5ng/ml) the protein levels of iNOS and Act1 were assessed by WesternBlot. (FIG. 11C) MSCs (Act1 knockdown or control) were treated withdifferent cytokines 12 hr, and iNOS expression was measured byquantitative RT-PCR. mRNA expression values are means±SEM. (FIG. 11D)MSCs (Act1 knockdown or control) were first treated with IFNγ and TNFαwith or without IL-17A (all cytokines supplemented at 5 ng/ml) for 12hr, and then cocultured with T cell hybridoma A1.1 cells at a ratio of1:10 for 12 hr. T cell proliferation was measured by ³H-Tdrincorporation, taking the proliferation level of A1.1 alone as 100%.

FIGS. 12A and 12B. (FIG. 12A). WT MSCs or auf1^(−/−) MSCs were treatedwith IFNγ and TNFα, or together with IL-17A (all cytokines supplementedat 10 ng/ml) for 12 hr, and iNOS, IL-6 and CXCL1 expression was measuredby quantitative RT-PCR. mRNA expression values are means±SEM of threewells from a representative of three independent experiments. (FIG.12B). WT MSCs were treated with IFNγ and TNFα (10 ng/ml), or togetherwith different concentrations of IL-17A; auf1^(−/−) MSCs were treatedwith IFNγ and TNFα (10 ng/ml), or together with 10 ng/ml of IL-17A.After 24 hr, cells were harvested for detection of iNOS by Western Blot.Western blot is a representative of three independent experiments.

FIGS. 13A and 13B. Wild-type (FIG. 13A) or auf1^(−/−) MSCs (FIG. 13B)were treated with IFNγ and TNFα, with or without IL-17A (all cytokinessupplemented at 10 ng/ml), for 6 hr, and then actinomycin D (5 g/ml) wasadded to stop transcription. At the indicated time points, mRNA levelswere assayed by quantitative RT-PCR, taking the expression level at thetime of actinomycin.D addition as 100%. mRNA expression values aremeans±SD of three wells from a representative of three independentexperiments.

FIGS. 14A and 14B. (FIG. 14A). WT MSCs or auf1^(−/−) MSCs were firsttreated with IFNγ and TNFα with or without IL-17A (all cytokinessupplemented at 5 ng/ml) for 12 hr, and then cocultured with T cellhybridoma A1.1 cells at a ratio of 1:10 for 12 hr. T cell proliferationwas measured by ³H-Tdr incorporation, taking the proliferation level ofA1.1 alone as 100%. (FIG. 14B). WT MSCs or auf1^(−/−) MSCs were firsttreated with IFNγ and TNFα with or without IL-17A (10 ng/ml), IFNγ andTNFα were supplemented at different cytokine concentrations, for 12 hr,and then cocultured with T cell hybridoma A1.1 cells at a ratio of 1:10for 12 hr. T cell proliferation was measured by ³H-Tdr incorporation,taking the proliferation level of A1.1 alone as 100%.

FIGS. 15A, 15B, 15C and 15D. (FIG. 15A). Serum levels of ALT weremeasured. (n=3-5 mice per group). (FIG. 15B). Calculation of absolutenumbers of mononuclear cells (MNCs) in liver tissues. (FIG. 15C).Absolute numbers of CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells were determined byFlow Cytometry. (FIG. 15D). H&E staining of liver sections at 8 h afterConA administration. a. Untreated mice; b. ConA+PBS; c. ConA+ wild-typeMSCs; d. ConA+IFNγ+TNFα pretreated wild-type MSCs; e.ConA+IFNγ+TNFα+IL-17A pretreated wild-type MSCs; f. ConA+auf1^(−/−)MSCs; g. ConA+IFNγ+TNFα pretreated auf1^(−/−) MSCs; h.ConA+IFNγ+TNFα+IL-17A pretreated auf1^(−/−) MSCs.

FIGS. 16A and 16B. Construction of an inducible mouse nitric oxidesynthase (iNOS) promoter-driven human indoleamine 2,3-dioxygenase (IDO)expression system in mesenchymal stem cells (MSCs). (FIG. 16A) Plasmidconstruction. (FIG. 16B) iNOS−/− MSCs, empty vector-transfected andhuman IDO-transfected iNOS−/− MSCs were stimulated with (+) and without(−) recombinant mouse inflammatory cytokines IFNγ and TNFα. The humanIDO expression was measured by western blotting.

FIGS. 17A, 17B, 17C and 17D. Type I interferons and FGF-2 down-regulatethe immunosuppressive effect of MSCs through attenuation NO production.(FIG. 17A) Type I IFN (IFN α) inhibited IFN γ+TNF α-induced iNOS proteinexpression in MSCs, without affecting the related transcriptionalfactors in STAT1 and NFκB pathways. (FIG. 17B) Supplement of type IIFNs: IFNα or β strikingly inhibited MSC-medicated immunosuppression inMSC+splenocyte+anti-CD3 system. (FIG. 17C) FGF-2 (FGF β) inhibited theNO production which was induced by IFN γ+TNF α or IFN γ+IL-β in MSCs,reflected by nitrate content in the culture supernatants. (FIG. 17D)supplement of FGF-2 significantly reduced the immunosuppressive effectof MSCs in MSC+splenocyte+anti-CD3 system.

FIGS. 18A, 18B, and 18C. (FIG. 18A) To determine the efficiency oftransduction, transduced cells were analyzed for GFP expression by flowcytometry. (FIG. 18B) MSC-GFP and MSC-IFNα were cultured at 5×10⁵ per mlfor 48 h. Supernatants were collected and IFNα concentration wasmeasured by IFNα Elisa Kit (PBL, NJ). (FIG. 18C) To test whether IFNαreleased by transduced cells had any biological functions, the surfaceexpression of H-2Kb on MSC-GFP, MSC-GFP treated with recombinant IFNα(ebiosience, CA) or supernatant of MSC-IFNα and MSC-IFNα was examined byflow cytometry after staining with APC-H-2Kb (ebiosience, CA).

FIGS. 19A, 19B, 19C, 19D, 19E and 19F. (FIG. 19A) 1×10⁶ B16 tumor cellswith or without 1×10⁶ MSC-GFP or MSC-IFNα were injected into C57BL/6mice intramuscularly. Twelve days later, tumors were excised andweighed. (FIG. 19B) 1×10⁶ B16 cells were intramuscularly with differentnumbers of MSC-IFNα: 1×10⁶ (1:1), 1×10⁵ (1:10), 1×10⁴ (1:100), 1×10³(1:1000), 1×102 (1:10000) or no MSC-IFNα. Twelve days later, tumors wereexcised and weighed. (FIG. 19C) 1×10⁶ B16 tumor cells with or withoutMSC-IFNα were injected into C57BL/6 mice intramuscularly. Mice survivalwas monitored for one hundred days after tumor inoculation. (FIGS. 19Dand 19E) 1×10⁶ B16 tumor cells were injected into C57BL/6 miceintramuscularly. After three (FIG. 19D) or four (FIG. 19E) days, 1×10⁶MSC-GFP or MSC-IFNαwas inoculated intramuscularly. Twelve days aftertumor inoculation, tumors were excised and weighed. (FIG. 19F) 1×10⁶ B16tumor cells were inoculated into C57BL/6 mice intramuscularly. Threedays later, PBS, 5 g recombinant IFNα or 1×10⁶ MSC-IFNα were injectedintramuscularly. After another nine days, tumors were excised andweighed. These experiments are repeated 2 to 3 times. Error bars,mean±s.d. for all plots. Statistical significance was assessed byunpaired two-tailed Student's t test.

FIGS. 20A, 20B and 20C. (FIG. 20A) 1×10⁶ luciferase labeled MSC-IFNαwere intramuscularly injected into C57BL/6 mice together with 1×10⁶ B16cells. At D0, D3, D7, D15 and D21 after injection, MSC were detected bylive imaging. Briefly, mice were anaesthetized, and 150 mg/kg ofD-luciferin (Caliper Lifescience, MA) was given intraperitonealy 15 minsbefore imaging. BLI data was acquired with Berthod NC100 imaging system.(FIG. 20B). Luciferase signal intensities were calculated and presented(Error bars, mean±s.d.). (FIG. 20C) 1×10⁶ B16 tumor cells with orwithout 1×10⁶ MSC-IFNα were injected into C57BL/6 mice intramuscularly.Twelve days later, tumors were 0 collected. For HE and Ki-67 (Abcam, MA)staining, tumor samples were fixed in 10% formalin at room temperaturefor one week and paraffin sections were prepared. For TUNEL assay,tumors were embedded in OCT, frozen immediately, and sections wereprepared for TUNEL assay with in situ cell death detection kit (Roche,Basel, Switzerland) following the producer's protocol.

FIGS. 21A, 21B, 21C, 21D, 21E, 21F and 21G. (FIG. 21A). 1 000 B16 cellswere seeded per well in 96-well plate with 0 ng/ml, 10 ng/ml or 100ng/ml recombinant mouse IFNα. Three days later, cells were incubated fortwo hours with 10 ul CCK8 (Dojindo, Shanghai, China), and O.D. (450 nm)was measured. (FIGS. 21B and 21C) 1×10⁶ B16 tumor cells with or without1×10⁶ MSC-GFP or MSC-IFNα were injected into C57BL/6 mice (FIG. 21B) orNOD-SCID (FIG. 21C) mice intramuscularly. Twelve days later, tumors wereexcised and weighed. (FIGS. 21D and 21E) 1×10⁶ B16 tumor cells with1×10⁵, 1×10⁴MSC-IFNα or no MSC-IFNα were injected into C57BL/6 mice(FIG. 21D) or NOD-SCID mice (FIG. 21E) intramuscularly. Twelve dayslater, tumors were excised and weighed. (FIG. 21F). 1×10⁶ B16 tumorcells with or without 1×10⁴ MSC-IFNα were injected into C57BL/6 mice. NKcells-specific depletion antibody anti-asialo GM (Wako, Osaka, Japan) orvehicle control were i.v injected every four days from the day beforetumor cell inoculation. Twelve days later, tumors were excised andweighed. (Figure G) 1×10⁶ B16 tumor cells with or without 1×10⁴ MSC-IFNαwere injected into C57BL/6 mice or β2m-deficient mice. Twelve dayslater, tumors were excised and weighed. These experiments are repeated 2to 3 times. Error bars, mean±s.d. for all plots. Statisticalsignificance was assessed by unpaired two-tailed Student's t test.

DETAILED DESCRIPTION OF INVENTION AND PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs and shall be understood to have themeanings described below. All publications and patents referred toherein are incorporated by reference in their entirety. In the case ofconflict, the present specification, including definitions, willcontrol. In addition, the materials, methods and examples areillustrative only and are not intended to be limiting.

As used herein, the term “about” will mean up to plus or minus 5% of theparticular term.

As used herein, the phrase “consisting essentially of” refers toexcluding other active ingredients or any other ingredient that canmaterially affect the basic characteristic of a composition, formulationor structure, but generally including excipients.

As used herein, invention an “effective amount” refers to that amount ofstem cells, cytokines, or a therapeutic composition containing both,that is sufficient to modulate, attenuate, or induce an immune response(i.e., suppression of T cell responses or promotion of an immuneresponse) in the subject thereby reducing at least one sign or symptomof the disease or disorder under treatment.

As used herein, the terms “treat,” “treating,” or “treatment” and thelike refers to alleviating signs or symptoms of the disease accomplishedby a administering a composition to a patient in need of such treatment.Such alleviation can occur prior to signs or symptoms of the diseaseappearing, as well as after their appearance, therefore it encompassesprophylactic and active treatment. In addition, “treat,” “treating” or“treatment” does not require complete alleviation of signs or symptoms,or a cure. At a cellular level it may include reduction of diseased ortarget cellular population by at least 10%, 25%, 50%, 75%, 80%, 85%,90%, 95%, or 99% as compared to untreated cells or cells treated withcontrol or a comparative agent.

As used herein, the terms “administration” or “administering” or“treatment regimen” within the scope of the present invention includes asingle therapeutic delivery, or multiple or repeated deliveries, or acontrol delivery therapeutic of any of the individual components of thepresent invention or in combination. Such terms are further meant toinclude modes of deliveries such as locally, systemically,intravascularly, intramuscularly, intra-peritoneally, inside theblood-brain barrier, organ-specific interventional injection or viaother various routes.

Generally speaking, the present invention describes composition,methods, and kits employing inflammatory cytokines such as IL-1 α,interleukin beta (IL-1 β), TNF α, IL-17 A, IFN-I, TGF β, FGF to pretreatMSCs to augment their immunomodulating effects such as immunosuppressiveor immune inducing effects, in prevention and treatment of variousdiseases such as multiple sclerosis, arthritis, lupus, sepsis,hepatitis, cirrhosis, Parkinson's disease, chronic infections, GvHD, andeven cancer and solid tumors.

Immunosuppression is elicited by inflammatory cytokine, produced duringan immune response. In the absence of inflammatory cytokines, MSCs donot gain their immunosuppressive properties. At least one aspect of thepresent invention describes the addition of inflammatory cytokines toprime and train MSCs for achieving a potent and long lasting inhibitoryfunction toward immune response. Such affect could manifest particularlyby the proliferation of activated T cells, or other immune responseparameters including_activated macrophages and other immune cells, serumlevels of inflammatory cytokines such as IFNγ or TNFα.

The crucial role of inflammatory cytokines has been by in vivo studieson graft-versus-host disease (GvHD), experimental autoimmuneencephalomyelitis, autoimmune hepatitis, chronic infections, livercirrhosis, lung cirrhosis, and rheumatoid arthritis. In at least oneaspect of the invention, genetically-modified MSCs are described thatcan reversely boost the immune response, owning to the secretion of alarge amount of chemokines and growth factors by MSCs in the absence orreduced NO or IDO. Therefore, the present invention offers powerfulsuppressive and augmentative strategies to control the immune response.

At least one aspect of the invention is directed to a population ofprimed or trained stem cells that are obtained by a process of (i)obtaining multipotent progenitor cells from a cell source, (ii)culturing said multipotent cells in a suitable medium, (iii) separatingmesenchymal stem cells from differentiated cells in said medium, (iv)activating at least a subset of said separated mesenchymal stem cellswith IFNγ and at least one cytokine in effective amounts selected fromthe group consisting of IL-1 α, IL-1 β, IL-17A, TGF α, FGF, IFN-I (IFNα, β), TNF α, and any combinations thereof. In one embodiment of thepresent invention, a subset of these trained stem cells produced by suchprocess enhance, boost, improve or induce immune response whenadministered to a subject in need thereof. In at least one embodiment,the subject is a mammal, preferably a human, or a human patientsuffering from a disease. In another embodiment, another subset oftrained stem cells are able to suppress, diminish or attenuate immuneresponse at a site of interest.

At least one aspect of the invention is a process of making a populationof primed or trained stem cells following the steps of (i) obtainingmultipotent progenitor cells from a cell source, (ii) culturing saidmultipotent cells in a suitable medium, (iii) separating mesenchymalstem cells from differentiated cells in said medium, (iv) activating atleast a subset of said separated mesenchymal stem cells with IFNγ and atleast one cytokine in effective amounts selected from the groupconsisting of IL-1 α, IL-1 β, IL-17A, TGF α, FGF, IFN-I (IFN α, β), TNFα, and any combinations thereof. In one embodiment of the presentinvention, the process employs a specific medium that can achieve theoptimal MSC properties. In another embodiment, the process includes afiltration or extraction step wherein all residual cytokines aresubstantially separated from the produced trained stem cells. Trainedstem cells used herein refers to the stem cells produced by the processdescribed herein and can consist of clonal, non-clonal or both types ofstem cells.

In one embodiment, subsets of trained stem cells are able to suppress,diminish or attenuate immune response at a site of interest. In anotherembodiment, the present invention describes pharmaceutical reagents thatblock the immunosuppressive properties of other treatment or biologicalregimens such as interferon or vaccines. In another embodiment, thepresent invention describes compositions that block immunosuppressiveproperties of tumor associated MSCs to enhance immunity toimmunosuppressive diseases such as cancer. Accordingly, MSC trainedcells can be adjunctive to or be use in combination with other standardtumor immune therapy protocols to boost immune response under stress.Such immune therapy can include vaccines and cancer immunotherapiesusing genetically, biologically and pharmaceutically-modified MSCs,vaccines, protein or gene therapies as immune adjuvants.

In another aspect of the invention, a method for stimulating immuneresponse is described in a subject in need thereof according to thesteps of (a) administering to the subject an effective amounts of acomposition containing an inhibitor to inducible nitric oxide synthase,an inhibitor to indoleamine 2, 3-dioxygenase, a population of induciblenitric oxide synthase (iNOS)-deficient mesenchymal stem cells, apopulation of indoleamine 2,3-dioxygenase (IDO)-deficient mesenchymalstem cells or any combinations thereof and (b) inhibiting the productionof one or more of nitrogen oxide (NO), indoleamine 2, 3 dioxygenase(IDO), or prostaglandin E 2 (PGE2). In one embodiment,

At least another aspect of the invention is directed to a compositionincluding (a) a population of isolated mesenchymal stem cells producedby a method comprising the steps of: (i) obtaining multipotentprogenitor cells from a cell source; (ii) culturing said multipotentcells in a medium to produce a subpopulation of mesenchymal stem cellsand a subpopulation of differentiated cells; (iii) separatingmesenchymal stem cells from differentiated cells in said medium, (iv)activating at least a subset of said separated mesenchymal stem cellswith IFNγ and at least one cytokine in effective amounts selected fromthe group consisting of IL-1 α, IL-1 β, TGF β, FGF, IFN-I (IFN α, β),TNF α, and any combinations thereof; and optionally (b) apharmaceutically acceptable carrier. In this aspect of the invention,the composition obtained induces the immune response of the subjectreceiving such composition. In another embodiment, such composition maybe substantially free of any cytokines used during the expanding phase.The term substantially free as used herein is meant to be have less than5%, 4%, 3%, 2%, 1%, 0.5%, 0.25% or 0.1% per weight of the composition.In another embodiment, the cell population may further contain cloned ornon-cloned mesenchymal stem cells, differentiated cells, or a mixturethereof.

In another embodiment, the activation step of the MSCs is accomplishedby presenting at least a subset of MSCs to IFNγ and at least onecytokine in effective amounts selected from the group consisting of IL-1α, IL-1 β, IL-17 A, TNF α, and any combinations thereof for sufficientperiod of time to illicit the desired immunosuppressive properties. Inthis embodiment, the composition obtained contains isolated MSCs thatsuppress or attenuate the immune response in the subject receiving suchcomposition both systemically or locally. In another embodiment, thecomposition is substantially free of any residual cytokines. In suchembodiment, the composition suppresses local immune T-cellproliferation. In another embodiment, the cell population may furthercontain cloned or non-cloned mesenchymal stem cells, differentiatedcells, or even a mixture thereof, wherein at least 50%, 60%, 70%, 75%,80% or 90% of said population of cells are made of cloned MSCs.

In another aspect of the present invention, the inventors describemethods for activating, enhancing, boosting or inducing immune responsein a patient in need thereof wherein a population of isolated MSCs areprimed or trained by exposure to (α) isolated IFNγ and (b) at least onecytokine in effective amounts selected from the group consisting of IL-1α, IL-1 β, TGF β, FGF, IFN-I (IFN α, β), TNF α, and any combinationsthereof for sufficient period of time. As used herein, the phrase“sufficient period of time” within the scope of the present inventionincludes a time period necessary to train the MSCs to exhibit thedesired properties. Such period of time ranges from at least 1 hour toabout 4 week, including 12 hours, 24 hours, 36 hours, 48 hours, 72 hoursand so on. In another embodiment, the cell population may furthercontain cloned or non-cloned mesenchymal stem cells, differentiatedcells, or even a mixture thereof, wherein at least 50%, 60%, 70%, 75%,80% or 90% of said population of cells are made of cloned MSCs.

In another embodiment, the population of isolated MSCs are administeredseparately or as a mixture with the isolated IFNγ and/or othercytokines. In at least another embodiment, the patient in need may besuffering from any one of an autoimmune disorder, allergy, sepsis,cirrhosis, cancer, viral infections and organ transplant.

In another aspect of the invention, the method of inducingimmunosuppression employs a population of trained mesenchymal stem cellsthat are obtained by a specific process of (i) obtaining multipotentprogenitor cells from a cell source such as a bone marrow, (ii)culturing such cells including the differentiated and multipotent stemcells in a suitable medium, (iii) separating mesenchymal stem cells fromdifferentiated cells in said medium, (iv) activating at least a subsetof said separated mesenchymal stem cells by exposing it for sufficientperiod of time to IFNγ and at least one cytokine selected from the groupconsisting of IL-1 α, IL-1 β, IL-17A, and TNF α. In at least oneembodiment, the medium used to activate the mesenchymal stem cells arefree of any other cytokine source.

In a preferred embodiment, the method of treating the subject in needincludes administering effective amounts of a composition containing thetrained mesenchymal cells locally to a site afflicted with a conditionfor treatment.

Another aspect of the present invention is directed to methods ofinducing the expression of NO synthases (iNOS), indoleamine2,3-dioxygenase (IDO) in at least a subset of said mesenchymal stemcells. In this aspect of the invention, increasing the concentration ofNO, IDO metabolites at the site of treatment improves the clinicaloutcome.

In another aspect of the present invention, a population of MSCs issuccessfully transduced to release functional IFN α. In at least oneembodiment, methods of using IFN α secreting MSC are described fortreating cancer and controlling tumor growth.

In yet another aspect of the present invention, populations of trainedstem cell are described in a therapeutic kit for use in a clinicalsetting. In at least one embodiment, the therapeutic kit furthercontains IFNγ and at least one cytokine such as IL-1 α, IL-1 β, IL-17A,IFN-I, TGF β, FGF, TNF α, and any combinations thereof. In particularembodiments, therapeutic kits may be assembled to be used forimmunosuppression or immune-enhancement with appropriate instruction totrigger such immune response respectively. In one embodiment, the kitmay consist essentially of trained MSCs, IFNγ and at least anothersecond cytokine, but free of any other active ingredients that wouldmaterially alter the behavior of the trained MSCs.

In one embodiment, the therapeutic kit for immunosuppression contains apopulation of trained stem cells, IFNγ and at least one cytokine such asIL-1 α, IL-1 β, IL-17A, TNF α, and any combinations thereof. In anotherembodiment, the therapeutic kit for immune enhancement contains apopulation of trained cloned stem cells, IFNγ and at least one cytokinesuch as IL-1 α, IL-1 β, IFN-I, TGF β, TNF α, and any combinationsthereof. In another embodiment, the cytokines are isolated type. Inanother embodiment, the instructions for using the kit articulate thesteps for triggering the desired clinical outcome.

In yet another embodiment, a method for stimulating immune response in apatient in need suffering for example from cancer or a viral infectionis described. In such embodiment, patients are administered effectiveamounts of a composition comprising an inhibitor to inducible nitricoxide synthase, an inhibitor to indoleamine 2, 3-dioxygenase, apopulation of inducible nitric oxide synthase (iNOS)-deficientmesenchymal stem cells, a population of indoleamine 2,3-dioxygenase(IDO)-deficient mesenchymal stem cells or any combinations thereof. In apreferred embodiment, the method cause inhibition of the production ofone or more of nitrogen oxide (NO), indoleamine 2, 3 dioxygenase (IDO),or prostaglandin E 2 (PGE2), 1-MT, 1400W, L-NMMA or other suitableagents. In this embodiment, the above mentioned inhibitors of iNOS orIDO are administered individually or as a mixture. In this aspect of theinvention, the patient's status is post receiving a regimen of immunetherapy including a regimen including the trained or primed MSCsdescribed herein, or another immune therapy regimen which can includetreatment with indicated interferons, antibody, cell therapy or othertherapies that modulate immune response.

Another aspect of the present invention describes methods for screeningreagents or drugs to inhibit or increase IDO activity in mammal MSCs,including human or mouse MSCs, by construction of human IDO-expressingmouse iNOS-deficient cells in which IDO protein consisting of the aminoacid sequence encoded by the human IDO gene under the control of mouseiNOS promoter, thereby improve the immunosuppressive function of MSCs.This aspect of the invention describes methods to screen reagents ordrugs to enhance or inhibit IDO activity in mouse model with human IDOexpression controlled by mouse iNOS promoter, such that saidadministration regulates IDO activity, thereby treat the diseaseinvolved in IDO abnormal expression in cancer or infections, especiallyin combination with immune therapies.

The human mesenchymal stem cells can be derived from a number of cellsource, for example, from placental derivatives or from bone marrow, orobtained from a number of different sources, including plugs of femoralhead cancellous bone pieces, obtained from patients with degenerativejoint disease during hip or knee replacement surgery, and from aspiratedmarrow obtained from normal donors and oncology patients who have marrowharvested for future bone marrow transplantation. Although the harvestedmarrow is generally prepared for cell culture separation by a number ofdifferent mechanical isolation processes depending upon the source ofthe harvested marrow (i.e., the presence of bone chips, peripheralblood, etc), the critical step involved in the isolation processes isthe use of a specially prepared medium that contains agents that allowfor not only mesenchymal stem cell growth without differentiation, butalso for the direct adherence of only the mesenchymal stem cells to theplastic or glass surface area of the culture dish.

By producing a medium that allows for the selective attachment andsurvival of the desired mesenchymal stem cells, which are present in themarrow samples in very minute amounts, it is possible to separate themesenchymal stem cells from the other cells (i.e., red and white bloodcells, fibroblasts, other differentiated mesenchymal cells, etc.)present in the bone marrow. Other sources of human MSCs includeumbilical cord, fat tissue and tooth root. MSC are multipotentprogenitors for a variety of cell types of mesenchymal cell lineage,including bone, cartilage, fat, tendon, nerve tissue, fibroblasts andmuscle cells. Mesenchymal stem cells can be isolated and purified formtissue such as bone marrow, blood (including peripheral blood),periosteum, and dermis, and other tissues which have mesodermal origins.In this regard, it has been found that although these progenitor cellsare normally present in bone marrow, for example, in very minute amountsand that these amounts greatly decrease with age (ie. From about1/10,000 cells in a relatively young patient to as few as 1/2,000,000 inan elderly patient), human mesenchymal stem cells can be isolated fromvarious tissues and purified when cultured in a specific medium by theirselective attachment, termed “adherence” to substrates.

Mesenchymal stem cells are typically identified based upon theexpression or lack of expression of particular markers. For example,MSCs are CD34−, CD11b, CD11c−, CD45−, MHC class II, CD44+, Sca-1+, andMHC class I low. In addition, MSCs can be identified by their ability todifferentiate into various mesenchymal cell types. In vitro experimentshave demonstrated that culture conditions, additives, growth factors andcytokines can precisely induce MSC to develop into a selectedmesenchymal cells. For example, dexamethasone in combination withisobutilmethylxanthine or insulin or a mixture ofisobutilmethylxanthine, insulin and indomethacin has been shown to pushthe MSCs toward differentiating into adipocytes. Similarly, MSCs candifferentiate into skeletal muscle cells when stimulated with5-azacytidine. 13-VGF has been shown to cause mesenchymal stem cells todifferentiate into cardiac muscle cells.

While the invention is not limited to the use of MSCs obtained by anyparticular method, MSCs can be isolated from bone marrow and umbilicalcord, purified and culturally expanded by any methodology acceptable inthe art. Plugs or aspirates of bone marrow cells (consistingpredominantly of red and white bold cells, and a very minute amount ofmesenchymal stem cells) are passed through syringes to dissociate thetissue into single cells. In a preferred embodiment a populationmultipotent progenitor cells are obtained from a suitable source such asbone marrow, umbilical cord or fat tissue, further cultured and expandedin a suitable medium typically containing glutamine. Then mesenchymalstem cells are identified and from differentiated cells and furtherexpanded in a medium containing IFNγ and at least one cytokine selectedfrom the group consisting of IL-1 α, IL-1 β, IL-17A, IFN-I, TGF β, FGF,TNF α, and any combinations thereof. In another embodiment, the clonalmesenchymal stem cells are identified and from differentiated cells andfurther expanded in a medium containing IFNγ and at least one cytokineselected from the group consisting of IL-1 α, IL-1 β, IL-17A, IFN-I, TGFβ, FGF, TNF α, and any combinations thereof. In either case, themeshenchymal stem cells expanded in such medium are trained andprogramed to suppress or enhance immune response in particular clinicalsetting.

In one embodiment, the multipotent progenitor cells are cultured insuitable medium such as complete medium (e.g., MEM medium with 10% fetalbovine serum) and humidified atmosphere. The media is not changed for atleast one day to allow the cells to attach to the culture dish.Thereafter the media is replaced every 3-4 days. When the cells havegrown to confluence, the cells are detached from the culture dish,preferably with trypsin. Cells can be subcultured in serum-free mediaafter removal or inactivation of the trypsin. Additional methods forisolating and culturing mesenchymal stem cells are provided in US PatentApplication Nos. 20070160583 and 20070128722 incorporated herein intheir entirety. MSCs can also be isolated from Wharton's jelly of theumbilical cord using similar methods.

In one embodiment, the isolated mesenchymal stem cells of this inventioncan be a subset of a heterogeneous cell population including certaindifferentiated cells. In another embodiment, the isolated mesenchymalstem cells are homogeneous composition containing only trained clonalMSCs. In another embodiment, the MSCs can be a mixed cell populationenriched in MSCs. In this regard, an isolated population of MSCs iscomposed of at least about 75% MSCs, or at least about 83%, 84%, 88%,89%, 90%, 91%, 93%, 95%, 96%, 97%, or 98% cloned MSCs, while the restcan include differentiated cells, progenitor cells, blood cells, or anyother suitable cells that would enhance the clinical outcome.

In effective amount refers to that amount of MSCs and cytokines that issufficient to attenuate an immune response (i.e., suppression of T cellresponses) in the subject thereby reducing at least one sign or symptomof the disease or disorder.

The mesenchymal stem cells used in accordance with the invention are, inorder of preference, autologous, allogenic or xenogeneic, and the choicecan largely depend on the urgency of the need for treatment

The cytokines of the present invention can be obtained by conventionalpurification methods, by recombinant technologies or from commercialsources. For example, the amino acid sequence of interferon-gamma (IFNγ)is provided under GENBANK Accession Nos. NP 000610 (human) and NP 032363(mouse). Commercial sources of IFN protein include, e.g., INTERMUNE(Brisbane, Calif.) and PeproTech, Inc. (Rocky Hill, N.J.). Likewise,tumor necrosis factor-alpha (TNFα, cachexin or cachectin) is providedunder GENBANK Accession Nos. NP 000585 (human) and NP 038721 (mouse) andcommercially available from sources such as ProSpec Bio (rehovot,Israel) and PeproTech, Inc. Similarly, human interleukin 1-alpha (IL1α)and interleukin 1-beta (IL1β) are known under Accession Nos. P01583 andP01584, respectively, and are available from commercial sources such asProSpec Bio and PeproTech, Inc. Interleukin 17A (IL17A) known underAccession Nos. BC067505 (human) and NM 010552 (mouse). When used inaccordance with this invention, the cytokines are “isolated”, i.e.,either homogenous (100%) or near homogenous (90 to 99%). In particularembodiments, the cytokines are recombinant proteins.

Interleukin 17A is one of the key inflammatory cytokines, primarilyproduced by IL-17 producing CD4⁺ T cells (Th17) cells which is awell-known cytokine for its proinflammatory functions in inflammatoryand autoimmune responses. IL-17A signals through a heteromeric receptorcomplex, IL-17RA and IL-17RC. Upon IL-17A binding, IL17RA recruits Act1, a critical downstream mediator of the IL-17A-induced signalingprocess. Although much is known about IL-17A-induced signaling pathwaysand the role of IL-17A in inflammatory and autoimmune diseases, itscellular targets and mode of action remain elusive.

The present invention employs IL-17A alone or in combination with othercytokines to facilitate trained MSCs that cause immune suppression. Theabove-referenced MSCs and cytokines can be in the form of a composition,e.g., a pharmaceutical composition suitable for administration to asubject in need of treatment with the same. The compositions of theinvention can be administered by any conventional method includingparenteral (e.g., subcutaneous or intramuscular) or intravenousinjection, intravenous infusion, specific organ intervention or topicalapplication. The treatment can be composed of a single dose or aplurality of doses over a period of time.

The pharmaceutical composition typically contains at least oneacceptable carrier. The carrier must be “acceptable” in the sense ofbeing compatible with the MSCs and cytokines and not deleterious to therecipients thereof. Typically, the carrier can be a suitable isotonicsolution such as phosphate-buffered saline, culture media, such as DMEM,physiological saline with or without albumin, 5% aqueous dextrose,and/or mixtures thereof, and other suitable liquids known to thoseskilled in the art.

In a preferred embodiment for use therapeutically, the pharmaceuticalcomposition of the invention can also be provided as a kit. A kit of theinvention can contain only a pharmaceutically acceptable carrier; anisolated population of mesenchymal stem cells stimulated or trained withisolated IFN γ isolated IL-1α; and isolated IL17A and furtherinstructions for using the kit in a method for attenuating an immuneresponse. In this aspect of the invention, the cells stimulated withcytokine components of the kit can be administered. The kit alsooptionally may include a means of administering the cells, for exampleby injection. In an optional embodiment, the compositions of thisinvention suitable for parenteral administration can further containantioxidant(s) in combination with one or morepharmaceutically-acceptable sterile isotonic aqueous or nonaqueoussolutions, suspensions or in the form of sterile lyophilized powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain the combination of theantioxidants, minerals and vitamins, buffers, solutes which render thefinal formulation isotonic.

The present invention further provides a composition comprising apopulation of isolated cloned MSCs, isolated IFNγ, isolated IL-1 α or β,and isolated IL-17A in admixture with a pharmaceutically acceptablecarrier. In another embodiment, the present invention provides acomposition comprising a population of isolated MSCs, isolated IFNγ,isolated TNF α, and isolated IL-17A in admixture with a pharmaceuticallyacceptable carrier. In an embodiment, the composition also comprisesisolated IL-1 α or β. The methods of use of such kits provide forattenuating an immune response following the steps of administering aneffective amount of MSCs, isolated IFNγ, isolated IL-1 α, TNF α, andisolated IL-17A to a subject in need of a treatment thereby attenuatingthe subject's immune response.

The present invention provides a method for attenuating an immuneresponse comprising administering an effective amount of isolatedmesenchymal stem cells, isolated IFNγ, isolated IL-1 α, and isolatedIL-17A to a subject in need of a treatment thereby attenuating thesubject's immune response. In an embodiment, the method furthercomprises isolated TNF-α.

In another embodiment, the treatment is directed towards multiplesclerosis, arthritis, lupus, sepsis, hepatitis, cirrhosis, Parkinson'sDisease, chronic infections and graft-versus-host disease. In anotherembodiment, the MSCs are provided as a pharmaceutical composition,wherein the MSCs are formulated with a cytokine cocktail prior toadministration. In another embodiment, the MSCs and cytokines areadministered as individual components. A subject in need of treatmentcan be a mammal (e.g., a human, monkey, cat, dog, horse, etc.) with aparticular disease or disorder associated with an adverse immuneresponse. In particular embodiments, the subject is human.

Effectiveness can also be determined by monitoring iNOS, IDO, and/orchemokine expression. Subjects benefiting from attenuation of an adverseimmune response include subjects having or suspected of having anautoimmune disorder (e.g., rheumatoid arthritis, diabetes mellitus type1, systemic lupus erythematosus, scleroderma, GvHD, cirrhosis orpsoriasis), allergy (e.g., hay fever), or sepsis. In addition, becauseinflammation orchestrates the microenvironment around tumors,contributing to proliferation, survival and migration, certain cancerpatients may also benefit from the present composition.

In organ transplant and bone marrow transplant, T cells of donor origincan recognize the recipient's MHC and lead to the development of GvHD.This often fatal disease is frequently unresponsive to variousimmunosuppressive therapies but new approaches targeting immunemodulatory molecules show great promise in treating GvHD. Most recently,MSCs have been shown to be highly effective in the treatment of GvHD inpre-clinical and clinical trials. The analysis presented herein furtherdemonstrates that MSC activity is mediated via the production of NO orIDO after stimulation with pro-inflammatory cytokines. Accordingly, thecomposition of this invention finds use in organ transplantation ortreatment of GvHD.

In vivo determination of suitable doses can be accomplished usingart-accepted animal models such as the DTH and GvHD models describedherein. However, as the present involves treatment under the care of aphysician or veterinarian, adjustments can be made to the amount andtiming of treatment during the course of treatment, based on theevaluation of the effectiveness of the treatment, which can vary fromsubject to subject. In addition, treatment can be provided at particularstages of immune responses in patients as described by the physician orveterinarian.

The present invention also provides a method for enhancing the efficacyof an immune therapy of cancer by administering to a subject receivingan immune therapy treatment and effective amount of an NOS and/or IDOinhibitor. In particular embodiments, the inhibitors are IDO andiNOS-selective inhibitors, e.g., as disclosed herein. An effectiveamount of such an inhibitor is an amount which provides at least a 50%,60%, 70%, 80%, 90%, 95%, or 97% decrease in the amount of NO productionand/or IDO activity upon administration of the immune therapy ascompared to a subject not receiving the inhibitors. In a particularembodiment, the method provides enhancing the therapeutic effectivenessof an interferon treatment (e.g. IFNγ) using an IDO and/oriNOS-selective inhibitor.

The present invention provides a method for modifying MSCs withinflammatory cytokines prior to administration into patients. Thismethod would dramatically enhance the efficacy of MSCs in clinicalsettings. In at least one aspect critical roles of iNOS and chemokinesin the immunosuppressive effect of MSCs, with the co-presence of IFNγand another cytokine, either TNFα, IL-1α or IL-1β as the requisite aredescribed. In another aspect, MSCs has been shown to switch to promoteimmune responses when inflammatory cytokines IFNγ and TNFα wereinadequate to induce sufficient immunosuppression.

At least in one embodiment, the role of IL-17A to change the dynamic ofthe interaction between MSCs and inflammatory cytokines is described.The present inventors have discovered that IL-17A enhances theimmunosuppressive function of MSCs, even in the presence of low dose ofinflammatory cytokines IFNγ and TNFα. Unlike its traditional role ofpromoting immune responses, as shown herein, IL-17A plays an importantrole in immunosuppression in the presence of MSCs. Thus, in certaincircumstances, blocking the activity of IL-17A can induce or enhanceimmune response. In at least one embodiment, the pathophysiologicalroles of IL-17A is described.

IL-17A is critical in promoting inflammation and autoimmunity. Those ofordinary skill in the art can appreciate that for the first time therole of IL-17A in enhancing immunosuppression in MSCs is substantiated.Previously, IL-17A have been widely reported to exacerbate diseaseprogress in multiple autoimmune diseases, including rheumatoid arthritis(RA), multiple sclerosis (MS) and inflammatory bowel disease (IBD), inwhich the IL-17A level is dramatically elevated. In addition, diseaseprogression slows down when IL-17A is genetically ablated or IL-17Ablocking antibody is administered.

However, IL-17A not always promotes immune responses, since past reportssuggest that IL-17A has a protective function in gut inflammatorydisorders. Genetic ablation or neutralization of IL-17A can actuallyaggravate disease progress in the dextran-sulphate-sodium (DSS) inducedcolitis model. In such context, those of ordinary skill in the art canappreciate that at least one aspect of the present invention providesthat IL-17A enhance the immunosuppressive property of MSCs. In at leastone embodiment, it is contemplated that MSCs may not suppress immuneresponses effectively without IL-17A.

In yet another aspect of the present invention, the inventorsdemonstrated a new function of IL-17A in enhancing immunosuppressionthrough a novel cell target, mesenchymal stem cells. Similarly it hasbeen shown that IL-17A exerts these effects by reversing the suppressionof gene expression conferred by mRNA decay factor AUF1.

In at least one embodiment of the present invention, Concanavalin A(“ConA”) induced liver injury in mice is employed for investigating thepathophysiological process of autoimmune or viral fulminant hepatitis,in which T cell responses play a pivotal role in mediating liver damage.As suppression of T cells responses can dramatically attenuate ConAinduced liver injury and adipose tissue derived stromal cells have beenshown to reduce ConA induced liver damage; the present inventors usedbone marrow derived MSCs and investigated the role of IL-17A inmodulating MSC-mediated treatment of liver injury. Thus, at least oneaspect of the present invention provide that IL-17A can dramaticallyenhance the immunosuppressive effects of MSCs.

The present invention further provides that MSCs can only marginallyaffect the progression of ConA-induced liver injury, because theimmunosuppressive capacity of MSCs requires stimulation by inflammatorycytokines. Although many cytokines can be produced after ConAadministration in vivo, these cytokines may only remain at high levelsfor a short time and not able to stimulate MSCs effectively whenadministered at a later time. Therefore, naive MSCs are not effective inattenuating ConA induced liver injury.

Accordingly, at least one aspect of the invention provides the new andnovel function of IL-17A in enhancing immunosuppression through a novelcell target, mesenchymal stem cells. It is further contemplated thatIL-17A may exert these effects by reversing the suppression of geneexpression conferred by mRNA decay factor AUF1. As described herein,IL-17A is a factor for enhancing MSC-mediated immunosuppression.

In certain cases, the inventors have found the need to control suchimmunosuppressive effect in vitro and in vivo, either positively ornegatively. In one embodiment the inventors screened the availablegrowth factors and cytokines, and found among them, there were twofactors strikingly down-regulating MSC-mediated immunosuppression: typeI interferons and fibroblast growth factor (FGF-2).

Type I interferons (IFNs), are a family of cytokines which render thehost immunity to eradicate viruses and other intracellular infections,whereas FGF-2 (FGF-β, basic fibroblast growth factor) belongs to afamily of genes encoding heparin-binding proteins with growth,antiapoptotic, and differentiation activity. However, no studies haverelated these two cytokines with regulation of immunosuppression. Type Iinterferons and fibroblast growth factor (FGF-2) serve as negativeregulators on MSC-mediated immunosuppression through down-regulation ofiNOS expression. As described herein, inventors found that these twofactors could potentially inhibit the immunosuppressive effect of MSCstowards T-cell proliferation (FIG. 16 ). Further analysis revealed that,supplement of either of these cytokines was able to strikingly reducethe expression of iNOS protein and NO production (FIG. 17 ).

The following non-limiting examples are provided to further illustratethe present invention.

EXAMPLES Example 1

Materials and Methods

Mice. Male C57BL/6, C3H/HeJCr and F1 (C57BL/6×C3H) mice, 6-8 weeks old,were from the National Cancer Institute (Frederick, Md.). IFNγ-R1^(−/−)mice and iNOS^(−/−) mice were from Jackson Laboratory (Bar Harbor, Me.).Mice were maintained in the Robert Wood Johnson Medical School Vivarium.Animals were matched for age and gender in each experiment, all approvedby the Institutional Animal Care and Use Committee.

Reagents. Recombinant mouse IFNγ and TNFα, IL-1α, and IL-1β monoclonalantibodies against mouse TNFα, IL-1 α, IL-1β, and CCR5, FITC-conjugatedanti-mouse CD11b, and PE-conjugated anti-mouse F4/80 were fromeBiosciences (La Jolla, Calif.). Recombinant mouse M-CSF and antibodiesagainst IL-1β and TGF-β. were from R&D Systems (Minneapolis, Minn.).Anti-IFNγ was from Harlan (Indianapolis, Ind.). Anti-CXCR3 was fromInvitrogen (Carlsbad, Calif.). Indomethacin, 1-methyl-DL-tryptophan(1-MT), and N G-monomethyl-L-arginine (L-NMMA) were from Sigma-Aldrich(St. Louis, Mo.).

Cells. MSCs were generated from bone marrow of tibia and femur of 6-10week old mice. Cells were cultured in a MEM medium supplemented with 10%FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin(all from Invitrogen). Non-adherent cells were removed after 24 hours,and adherent cells were maintained with medium replenishment every threedays. To obtain MSC clones, cells at confluence were harvested andseeded into 96-well plates by limited dilution. Individual clones werethen picked and expanded. Cells were used at 5th to 20th passage.

T cell blasts were generated from CD4⁺ T cells purified by negativeselection with CD4^(−/−) T cell subset isolation kits (R&D Systems).Cells (1×10⁶ cells/ml) were activated by plastic-bound anti-CD3 andsoluble anti-CD 28 for 48 hours, then cultured with IL-2 (200 U/ml)alone for 48 hours. All T cell cultures were maintained in RPMI-1640medium supplemented with 10% heat-inactivated FBS, 2 mM glutamine, 100U/ml penicillin, 100 μg/ml streptomycin, and 50 mMP-ME (completemedium).

Activated splenocyte supernatant was harvested from 48 hour-cultures ofsplenocytes (2×10⁶/ml) activated by plastic-bound anti-CD3, thenfiltered with a 0.1 μm filter and frozen.

Detection of Cytokines, Chemokines, and NO. Culture supernatants wereassayed for 20 different cytokines and chemokines with a multiplex beadarray kit (Invitrogen, Carlsbad, Calif.) using Luminex Technology(Bio-Plex System, Bio-Rad, Hercules, Calif.). IFNγ was assayed by ELISA(BD Biosciences, San Jose, Calif.). NO was detected using a modifiedGriess reagent (Sigma-Aldrich). Briefly, all N03 was converted into N02by nitrate reductase, and total NO2 detected by the Griess reaction(Miranda, et al. (2001) Nitric Oxide 5:62-71).

Real-Time PCR. RNA was isolated from cell pellets using an RNEASY MiniKit. First-strand cDNA synthesis was performed using SENSISCRIPT RT Kitwith random hexamer primers (all kits from Qiagen, Valencia, Calif.).mRNA of the genes of interest were quantified by real-time PCR (MX-4000from Stratagene, La Jolla, Calif.) using SYBR Green Master Mix (AppliedBiosystems, Foster City, Calif.). Total amount of mRNA was normalized toendogenous .beta.-actin mRNA. Primers sequences for iNOS were: forward,5′-CAG CTG GGC TGT ACA AAC CTT-3′ (SEQ ID NO:1); reverse, 5′-CAT TGG AAGTGA AGC GTT TCG-3′ (SEQ ID NO:2). Other primers were from the RT2PROFILER™ PCR Array Mouse Chemokines & Receptors kit (Superarray,Frederick, Md.).

Chemotaxis Assay. Chemotaxis was tested with the NeuroProbe CHEMOTXChemotaxis System (NeuroProbe, Gaithersburg, Md.), as described (Shi, etal. (1993) J. Immunol. Meth. 164:149-154). The lower chambers of the96-well plate were filled with supernatant from MSCs stimulated withIFNγ plus TNFα (20 ng/ml each or Sup.CD3-act (1:2 dilution). Apolyvinylpyrrolidine-free polycarbonate membrane with 5 μm pores wasthen overlaid. T cell blasts (1.25×10⁵) were added to the top chambers.After a 3-hour incubation, cells that had migrated through pores andinto bottom wells were quantified using MTT assay (Shi, et al. (1993)supra). A chemotaxis index was calculated as the ratio of the number ofT cell blasts migrated in response to MSCs compared to the numbermigrating to medium alone.

The immunosuppression resulting from T cell migration towardinflammatory cytokine-activated MSCs was examined in a similar set-up.MSCs (2×10⁴) were added to the lower chamber with or without stimulationwith IFNγ and TNFα (20 ng/ml each) for 24 hours. Activated T cell blastswere then added to the upper chamber, as above. IL-2 was added to bothchambers. After 3 hours, both chambers were pulsed with 3H-thymidine,and cell proliferation assessed 6 hours later.

GvHD Induction and Modulation by MSCs. C57BL/6×C3H F1 mice at 8-weeksold were lethally irradiated (13 Gy) and after 24 hours were infused bytail vein injection with nucleated bone marrow cells (5×10⁶) andsplenocytes (5×10⁶) isolated from C57BL/6 parent mice. On days 3 and 7following bone marrow transplantation, the recipients were administratedwith 0.5×10⁶MSCs derived from C57BL/6 wild-type, IFNγR1^(−/−), oriNOS^(−/−) mice via the tail vein. Some wild-type MSC groups were alsoinjected i.p. with the iNOS inhibitor, NG-monomethyl L-arginine (L-NMMA,500 μg/mouse), anti-IFNγ (400 μg/mouse), or a cocktail of threeantibodies against TNFα, IL-1α and IL-1β (200 μg each/mouse) daily for 7days starting immediately after the first MSC administration. Asnegative controls, the F1 mice were injected with F1 bone marrow cells.Mice were observed daily for GvHD signs (wasting, ruffled hair, andhunched back) and euthanized upon becoming moribund, thus markingsurvival time. On day 14, various tissues were collected and 5 mparaffin sections prepared and stained with hematoxylin/eosin (H&E).

Induction of DTH Response and Histology Analysis. C57BL/6 mice (6-8weeks old) were immunized by tail base injection of ovalbumin (OVA, 10μg in 50 μl saline) emulsified with 50 μl complete Freund's adjuvant.DTH was tested after 5 days, by challenging with 200 μg aggregated OVAin 30 μl saline injected into the right hind footpad. The left footpadwas injected with 30 μl of saline as a negative control. After 24 hours,antigen-induced footpad thickness increment was measured using a caliperand calculated as: (Rimm-Limm)-(R.unimm-L.unimm), where R and L arethickness of right and left footpads.

Statistical Analysis. Significance was assessed by unpaired two-tailedStudent's t-test or analysis of variance (ANOVA).

Example 2

Immunosuppressive Function of MSCs is Induced by ProinflammatoryCytokines.

To identify the underlying mechanisms, clones of mouse MSCs wereemployed. The stem cell characteristics of these clones were defined bytheir ability to differentiate into adipocytes or osteoblasts and bytheir expression of surface markers: CD34; CD11b; CD11c; CD45; MHC classII; CD44⁺; Sca-1⁺; MHC class I1^(low). All results presented herein werereplicated using at least three different MSC clones.

Since most reported studies of immunosuppression by MSCs are based ontheir effects on T cell proliferation and cytokine production, theeffect of MSCs was first examined on the IL-2-driven proliferation of Tcell blasts. Fresh CD4⁺ T cell blasts were generated from splenocytes byactivation with anti-CD3 followed by expansion with IL-2 for severaldays (Devadas, et al. (2006) Immunity 25:237-247; Radvanyi, et al.(1996) Cell Immunol. 170:260-273). T cell blasts were added at a 1:20ratio (MSC:T cells) along with IL-2 (200 U/ml). Cell proliferation wasassessed by ³H-Tdr incorporation after 8 hours. Surprisingly, it wasfound that the IL-2 driven proliferation of these T cell blasts wasunaffected by the addition of MSCs. MSCs also had no effect on theproliferation of T hybridoma A1.1 cells. These T cells blasts and Thybridoma cells, however, produce no cytokines unless reactivatedthrough the TCR (Fotedar, et al. (1985) J. Immunol. 135(5):3028-33).Thus, in the absence of T cell cytokines, MSCs were unable to suppress Tcell proliferation.

To examine the possibility that cytokines induce the immunosuppressivecapacity of MSCs, these culture conditions were reproduced by combiningMSCs and fresh splenocytes at graded ratios in the presence of anti-CD3.The results of this analysis indicated that T cell proliferation wascompletely blocked when MSCs were added at a ratio as low as 1:60 (MSCto splenocyte). Importantly, to exert their immunosuppressive effect,MSCs do not have to be syngeneic. A similar effect was found on purifiedCD4⁺ or CD8⁺ T cells activated by plastic-bound anti-CD3 antibody andanti-CD28 using MSCs from between the 5th and 20th passage. Thus, underconditions in which MSCs and T cells are in co-culture during T cellactivation, the resultant T cell response was strongly suppressed byMSCs, indicating that T cell-produced cytokines may have a role. Theimmunosuppressive capacity of MSC clones generated from different mousestrains was also examined. It was observed that those clones thatexhibited better differentiation potential had a greater capacity forimmunosuppression.

To determine whether cytokines secreted by activated T cells areresponsible for the induction of immunosuppression by MSCs, mixedco-cultures of MSCs with T cell blasts (as described above) weresupplemented with supernatant from a culture of anti-CD3-activatedsplenocytes. The resultant T cell proliferation was greatly inhibited.It was also observed that the proliferation of A1.1 cells in co-culturewith MSCs was inhibited by supplementation with the activated splenocytesupernatant. These experiments indicate that some product(s) ofactivated T cells is required to induce immunosuppression by MSCs. Toidentify the culpable cytokine(s), the activated splenocyte supernatantwas treated with neutralizing antibodies against various cytokinesbefore addition to the co-cultures. This analysis indicated thatneutralization of IFNγ completely reversed the inhibition ofproliferation of T cell blasts co-cultured with MSCs supplemented withthe anti-CD3 activated splenocyte supernatant. These results implicateIFNγ as a key cytokine in this process, and reveal that under certainconditions this major proinflammatory cytokine can instead mediateimmunosuppression.

The effect of IFNγ was then tested directly by adding isolatedrecombinant IFNγ (20 ng/ml) instead of activated splenocyte supernatantto the mixed co-cultures of MSC+ T cell blasts or MSC+A1.1 cells.Surprisingly, IFNγ alone did not induce immunosuppression. Several otherproinflammatory cytokines were then added (20 ng/ml each) and it wasfound that concomitant addition of either TNF α, IL-1α, or IL-1β alongwith IFNγ was required to achieve suppression of T cell proliferation inco-cultures with MSCs (1:20 ration MSC:T cells) (FIG. 1 ). Therefore,induction of the immunosuppressive function of MSCs byanti-CD3-activated splenocyte supernatant may be due to IFNγ acting inconcert with either TNFα; IL-1α; or IL-1β on the MSCs. Thus, while IFNγis absolutely required, this cytokine alone is not sufficient; properimmunosuppression signaling in MSCs requires the concerted action ofIFNγ and any of the other three cytokines.

Neutralizing antibodies against TNFα; IL-1α; or IL-1β, individually ortogether, were added to the activated splenocyte supernatant beforeaddition to mixed co-cultures of MSCs and T cell blasts. Whileindividual antibodies had no effect, simultaneous blockade of all threecytokines completely reversed the inhibition of T cell proliferation.Other cytokines, such as GM-CSF (Granulocyte-macrophagecolony-stimulating factor) and IL-6 (Interleukin-6), had no effect. Thedata herein indicate that the combination of the IFN with any of theother three proinflammatory cytokines, TNFα, IL-1α or IL-1β is fullyresponsible for inducing the ability of MSCs to inhibit T cellproliferation, and that TNFα, IL-I α, and IL-I β are interchangeable inacting together with IFNγ.

It is contemplated that MSCs must encounter some level of IFNγ arisingfrom initial T cell activation. Indeed, it was found that MSCs do notaffect the initial T cell response when present during theiranti-CD3-induced activation, as demonstrated by normal increases in CD69expression. As further evidence that IFNγ released from splenocytesafter initial activation was key to inducing immunosuppression by MSCs,it was observed that MSCs derived from mice deficient in IFNγ receptor 1(IFNγ R1^(−/−)) were incapable of immunosuppression. Several clones ofthese IFNγ R1^(−/−) MSCs were derived (all capable of differentiationinto adipocytes and osteoblast-like cells), and none of the five clonestested were able to suppress anti-CD3-induced splenocyte proliferation,supporting the understanding that IFNγ is essential in the induction ofthe immunosuppressive function of MSCs.

These results indicate that the initial production of IFNγ and othercytokines by cells in close proximity to MSCs are critical to induce theimmunosuppressive capacity. Indeed, anti-IFNγ (20 μg/ml) also completelyblocked the suppressive effect of MSCs in this setting. In addition,although antibodies against TNFα, IL-1α, and IL-1β (20 μg/ml each) wereineffective individually, immunosuppression was prevented when all threeantibodies were added together, similar to their effect when added toactivated splenocyte supernatant. Therefore, the concomitant action oflocally produced IFNγ along with TNFα, IL-1α, and IL-1β is sufficient toinduce MSCs to become immunosuppressive.

Example 3

Immunosuppression by MSCs Requires Nitric Oxide

To identify the mechanism through which immunosuppression bycytokine-exposed MSCs is effected, the response of anti-CD3-activatedsplenocytes co-cultured with MSCs (1:20, MSC:splenocytes) in a TRANSWELLsystem was examined in various configurations. When separated by apermeable membrane (0.4 μm pore membrane) in the two chambers of thewell, MSCs had almost no effect on T cell proliferation, indicating thata cell membrane-associated protein or other local acting factor(s) wascritical for the suppression of T cell proliferation by cytokine-primedMSCs. While a recent report (Sato, et al. (2007) supra) showed thatPGE-2, but not IDO, is required, it was found that PGE-2 was notinvolved. In fact, no effect was found on immunosuppression by MSCs byindomethacin (10 μM, a PGE-2 blocker), anti-IL-1β (20 μg/ml), anti-TGFβ(20 μg/ml) or 1-methyl-DL-tryptophan (1-MT, 1 mM, an IDO inhibitor),thereby ruling out these factors.

Nitric oxide (NO) at high concentrations is known to inhibit T cellresponses. It diffuses quickly from its source, but the concentration ofthe active form drops off within about 100 μm. Therefore, NO can actonly in close proximity to the cells producing it, which is consistentwith the predicted characteristics of the factor that mediatesimmunosuppression by MSCs. To determine whether NO had such a role, itsproduction was shut down using a selective inhibitor of iNOS activity, NG-monomethyl-L-arginin (L-NMMA). When added to mixed co-cultures of MSCsand splenocytes in the presence of anti-CD3, L-NMMA completely restorednormal splenocyte proliferation. Other iNOS inhibitors such as 1400 Wand L-NAME showed the same effect. Furthermore, MSCs derived from micedeficient in iNOS (iNOS^(−/−)) had almost no effect on splenocyteproliferation. In addition, of five clones of iNOS^(−/−) MSCs derived(all capable of differentiation into adipocytes and osteoblast-likecells), none were immunosuppressive. These results indicate that theactivity of NO produced by MSCs in response to cytokine-inductionmediates their suppression of T cell responses.

The analysis herein indicates that immunosuppression by MSCs is inducedby IFNγ and proinflammatory cytokines and is mediated through NO.Accordingly, it was contemplated that MSCs could upregulate theirexpression of iNOS and produce NO after exposure to these cytokines. Toexamine this, MSCs were treated with activated splenocyte supernatantand the level of iNOS mRNA assayed by real-time PCR and compared toβ-actin. The results of this analysis indicated that iNOS wassignificantly upregulated in MSCs by 4 hours after stimulation, withhigh-level expression sustained for at least 48 hours. At 12 hourspost-stimulation, the level of iNOS mRNA was more than 7 times greaterthan β actin message, indicative of extremely high expression. A similareffect was observed when IFNγ and TNFα (20 ng/ml each) were addedtogether, while either alone was ineffective.

In addition, IL-1α and IL-1β were again interchangeable with TNFα inthis regard. When antibodies were added to neutralize cytokineactivities in anti-CD3-activated splenocyte supernatant, it was observedthat anti-IFNγ alone, or the 3-antibody combination against TNFα, IL-1α,and IL-1β, prevented iNOS upregulation by MSCs. When antibodies againstTNFα, IL-1α or IL-1β were used singly or doubly, there was no effect.Therefore, the same cytokines that induce immunosuppression are alsopotent inducers of iNOS expression by MSCs.

To determine whether iNOS expression in cytokine-treated MSCs indeedleads to NO production, two stable breakdown products of NO, nitrate(NO3) and nitrite (NO2), were measured in conditioned medium from MSCstreated with anti-CD3-activated splenocyte supernatant. The amount ofNO2 produced by MSCs after treatment was at least 10 times greater thanthat from similarly treated CD11b⁺F4/80⁺ macrophages, which are known tobe abundant producers of NO. These results are consistent with the highlevels of iNOS mRNA expression described herein. Thus, upregulation ofiNOS expression by MSCs in response to proinflammatory cytokines leadsto production of NO, which can act on T cells in close proximity.

In the present study, with T cell activation or when exogenousinflammatory cytokines are added, the T cells first enter cell cyclearrest and then die within 24 hours. It was also observed that thisapoptosis was dependent on NO, since T cell apoptosis was not observedwhen iNOS inhibitors were used. Apoptosis was also absent wheniNOS^(−/−) or IFNγ R1^(−/−) MSCs were used. Therefore, NO-induced cellcycle arrest and apoptosis of T cells are part of the mechanism ofimmunosuppression mediated by inflammatory cytokine-activated MSCs.Differences between species in inflammatory cytokine-induced expressionof iNOS has been noted in macrophages (Schneemann & Schoedon. 2002) Nat.Immunol. 3(2):102). NO was found to be induced by inflammatory cytokinesin macrophages of mouse, rat, and bovine origin, but not caprine, lapin,porcine, and human macrophages (Schneemann & Schoedon (2002) supra;Jungi, et al. (1996) Vet. Immunol. Immunopathol. 54:323-330). Thus, theroles of IDO and NO in the inhibition of T cell proliferation by MSCsfrom mouse and human were analyzed in a side-by-side comparison. It wasfound that inhibition of NO by L-NMMA completely reversedimmunosuppression by mouse MSCs, whereas the inhibition of peripheralblood mononuclear cell proliferation by human MSCs was reversed by 1-MT,indicating that MSCs from humans utilize IDO as the major effector ofimmunosuppression, in comparison to mouse MSCs which utilize NO (Ren G,Su J, Zhang L, Zhao X, Ling W, L'huillie A, Zhang J, Lu Y, Roberts A I,Ji W, Rabson A B, Shi Y. Species variation in the mechanisms ofmesenchymal stem cell-mediated immunosuppression. Stem Cells 2009,27:1954-1962).

Example 4

Chemoattractive Property of MSCs is Induced by Proinflammatory Cytokines

In several studies, effective immunosuppression by MSCs in vivo has beenachieved with as few as one to five MSCs per million somatic cells andoften endures for months, with complete cure of immune disorders in someinstances. Considering that MSCs are immobile after settling in tissues,and that immunosuppression is mediated by NO, which acts only verylocally near its source, this immunosuppressive effect is astonishing.It was contemplated that cytokine-induced MSCs might have a mechanism toattract immune cells to their vicinity, where the locally highconcentrations of NO could act effectively on the target T cells. Toexplore this, co-cultures of MSCs and splenocytes were monitored overtime under the microscope.

Upon anti-CD3-stimulation, the splenocytes were observed to activelymigrate toward the spindle-shaped MSCs. In contrast, no migrationoccurred in the absence of anti-CD3 stimulation. Since splenocytes havelimited viability, the lack of locomotion toward MSCs in the absence ofstimulation might be due to the poor health of these cells in vitro. Toexclude this, activated-splenocyte-supernatant-primed MSCs were examinedfor their ability to attract A1.1 T hybridoma cells, which survive welleven in the absence of IL-2. Under these conditions, time-lapsemicrovideography revealed brisk migration of T cells toward MSCs within1.5 hours of co-culture initiation. Without priming of MSCs, however,there was no net movement of T cells toward the MSCs. Therefore, MSCspromote the migration of T cells only after MSCs having been exposed toproinflammatory cytokines.

To examine the role of various cytokines in enabling MSCs to attract Tcells, MSCs were pretreated with various combinations of recombinantcytokines and the resultant migration of pre-activated T cells inco-cultures was observed. This analysis indicated that the same T cellcytokine pairs (i.e., IFNγ and TNFα; IFNγ and IL-1α or IFNγ and IL-1β)that had induced the immunosuppressive function of MSCs also caused themto attract T cells. Likewise, using antibody neutralization of specificcytokines, it was found that migration toward MSCs was prevented byanti-IFNγ alone, or by blocking TNFα, IL-1α and IL-1β as a threesome,identical to their effects on activated-splenocyte-supernatant-inducedMSC suppression of T cell proliferation. Therefore, the cytokine-inducedimmunosuppressive function of MSCs is likely to depend on the migrationof lymphocytes into proximity with MSCs, where NO levels are highest.

Example 5

Proinflammatory Cytokines Induce MSCs to Produce Chemokines that areCritical for Immunosuppression

The robust migration of activated T cells toward cytokine-primed MSCsindicated that the MSCs secrete potent chemoattractants, such aschemokines. Accordingly, the production of leukocyte chemokines by MSCscultured under various conditions was determined by assaying thesupernatant. No significant chemokine production was observed for MSCscultured alone without cytokines, corroborating the findings that MSCsin their innate form are unable to attract T cells. When co-culturedwith anti-CD3-activated splenocytes, however, MSCs produced severalchemokines in large amounts, including CXCL-9 (MIG) at 1.5 ng/ml (12ng/ml in another experiment) and CXCL-10 (IP-10) at 50 ng/ml at a MSC:splenocyte ratio of 1:60. These are potent T cell-specific chemokines;it has been shown that concentrations of only 1 to 10 ng/ml of eitherchemokine alone drive significant chemotaxis in vitro (Loetscher, et al.(1998) Eur. J. Immunol. 28:3696-3705; Meyer, et al. (2001) Eur. J.Immunol. 31:2521-2527). The production of CXCL-9 and CXCL-10 wasinhibited by antibody neutralization of IFNγ alone, or all threecytokines TNFα; IL-1.α, and IL-1β, similar to the effects onimmunosuppression induction. Chemokine production was similarly inducedby adding recombinant IFNγ and TNFα (20 ng/ml each) to MSCs alone, withTNFα again being interchangeable with IL-1α and IL-1β. Therefore, thesecytokines are sufficient to induce MSC expression of chemokines, whichare likely to be responsible for driving T cell chemotaxis toward MSCs.Thus, once they have migrated into close proximity with MSCs, activatedT cells would be expected to secrete cytokines that induce theproduction of additional chemokines by the MSCs, thus creating apositive feedback loop to attract still more T cells to the vicinity ofMSCs.

To systematically examine the chemokine expression profile of MSCs, theexpression of 84 different genes encoding chemokines and their receptorswas examined in MSCs treated with supernatant from naive oranti-CD3-activated splenocytes. Total RNA was analyzed by real-time PCRusing the Mouse Chemokines and Receptors RT2 PROFILER® PCR Array kit,and chemokine mRNA levels compared to that of β actin (Table 1). Thesome human cytokine combination also induced similar chemkine productionin human MSCs.

TABLE 1 Induction of Expression of Chemokines and Related Genes in MSCsTreated with Supernatant from activated T cells (β-actin defined as 1 ×10⁷ units) Gene Naive Activated Fold Symbol Description Spin Sup SpinSup Increase Cxcl9 Chemokine (C-X-C motif) ligand 9, MIG 4 8,963,2942,025,082 Cxcl5 Chemokine (C-X-C motif) ligand 5, ENA- 2 4,302,8671,978,890 78 Cxcl2 Chemokine (C-X-C motif) ligand 2, Groβ 2 2,711,8381,681,250 Ccl7 Chemokine (C-C motif) ligand 7. MCP-3 0 24,269 1,111,786Cxcl10 Chemokine (C-X-C motif) ligand 10, IP- 111 19,719,159 177,864 10Cxc11 Chemokine (C-X-C motif) ligand 1, Gro α 47 5,170,437 110,278 Ccl5Chemokine (C-C motif) ligand 5, 215 8,022,162 37,344 RANTES Ccl2Chemokine (C-C motif) ligand 2, MCP-1 3,252 11,653,869 3,584 Cxcl11Chemokine (C-X-C motif) ligand 11, 5 17,370 3,534 ITAC Ccrl2 Chemokine(C-C motif) receptor-like 2 110 56,765 518 Ccl17 Chemokine (C-C motif)ligand 17, TARC 294 23,212 79 Cx3cl1 Chemokine (C-X3-C motif) ligand 1,69,309 2,349,699 34 fractalkine Cmkor1 Chemokine orphan receptor 132,965 617,300 19 Ccl8 Chemokine (C-C motif) ligand 8, MCP-2 628 10,07016 Ccl9 Chemokine (C-C motif) ligand 9 182 2,193 12 Ccr9 Chemokine (C-Cmotif) receptor 9 999 3,860 4 Cxcl13 Chemokine (C-X-C motif) ligand 13,19,067 48,073 3 BCA-1 Cxcr6 Chemokine (C-X-C motif) receptor 6 4,9437,516 2 Cmklr1 Chemokine-like receptor 1 49,839 47,340 1 Ccbp2 Chemokinebinding protein 2 0 9,015 N/A Actb β-actin 10,000,000 10,000,000 1 MSCs(1 × 10⁶/T-25 flask in 5 ml of complete medium) was stimulated withsupernatant from naïve or activated T cells (50% of the final volume)for 12 hr. Chemokine and chemokine receptor gene expression were assayedby real-time PCR.

It was found that, except for low levels of CX3CL-1 (fractalkine) andCXCL13 (Chemokine (C—X—C) ligand 13, BCA-1), mRNA levels in MSCs exposedto naive splenocyte supernatant were insignificant. Strikingly,treatment of MSCs with activated splenocyte supernatant resulted in amore than one million-fold increase in some chemokines, such as CXCL2(Chemokine (C—X—C) ligand 2, Grop), CXCL5 (Chemokine (C—C) ligand 5,RANTES), CXCL9 (Chemokine (C—X—C) ligand 9, MIG), CXCL10 (Chemokine(C—X—C) ligand 10, IP-10) and CCL7 (Chemokine (C—C) ligand 7, MCP-3). Inabsolute terms, some chemokines reached the same level of expression asβ-actin, or even higher. For example, CXCL10 showed twice the mRNA copynumber as β-actin. The chemokines that were most highly induced areextremely potent inducers of leukocyte chemotaxis and are likely to playan important role in immunosuppression by MSCs. In fact, it was observedthat antibody blockade of CXCR3, a receptor for the T cell chemokinesCXCL9, CXCL10 and CXCL11 (Lazzeri & Romagnani, (2005) Curr. Drug TargetsImmune Endocr. Metabol. Disord. 5:109-118), which were all highlyinduced in MSCs, inhibited the chemotaxis of T cell blasts toward MSCsand reverted the suppression of their proliferation.

To directly examine the chemotaxis-driving capacity of proinflammatorycytokine-induced MSC supernatant, the CHEMOTX Chemotaxis System(NeuroProbe) was employed. This system is composed of upper and lowerchambers separated by a polyvinylpyrrolidine-free polycarbonate membrane(5 μm pore size). Supernatant from MSC cultures was placed in the lowerchambers and activated CD4⁺ or CD8⁺ T cell blasts were added to theupper chambers in the presence of IL-2. Chemotaxis was quantified after3 hours. It was found that dramatic chemotaxis by both CD4⁺ and CD8⁺ Tcells occurred in response to culture supernatant from MSCs treated withIFNγ plus TNFα or with IFNγ plus IL-1. Similar results were obtainedwith supernatant from MSCs treated with medium conditioned by activatedsplenocytes.

In contrast, negative control supernatants from untreated MSCs oractivated splenocytes alone were non-chemotactic, as was the directaddition of IFNγ plus TNFα without MSCs. Importantly; this chemotacticactivity could be blocked by antibodies against CXCR3 and CCR5, two ofthe most important T cell-specific chemokine receptors, especially whenboth antibodies were added together. In addition to recruiting T cells,cytokine-activated MSCs also attracted bone marrow-derived dendriticcells, macrophages, and B cells.

The CHEMOTX system was also used to examine the role of chemotaxis inthe inhibition of T cell proliferation. In this assay, MSCs were addedto the lower wells, with or without addition of IFNγ plus TNFα, and Tcell blasts (with IL-2) were added to the upper wells. In this set-up,chemokines produced by MSCs in the lower wells should induce T cellmigration through the membrane and into the lower wells, where NOproduced by MSCs could thus inhibit their proliferation. After a 3-hourincubation, both the upper and lower wells were pulsed with 3H-thymidinefor an additional 6 hours and cells in both wells harvested fordetermination of proliferation. Proliferation levels of both CD4⁺ andCD8⁺ T cell blasts were significantly inhibited by MSCs in the presenceof IFNγ and TNFα. Again, blocking antibodies against the T cellchemokine receptors, CXCR3 and CCR5, significantly reversed this effect.These data further indicate that T cell chemotaxis is critical inMSC-mediated immunosuppression.

Taken together, these results indicate that when MSCs are exposed topro-inflammatory cytokines during an immune reaction, they produce largeamounts of several chemokines, especially those specific for T cells,which thus attract T cells into close proximity to MSCs, where highconcentrations of NO act to suppress T cell function.

Example 6

Prevention of Delayed-Type Hypersensitivity (DTH) and Graft-Versus-HostDisease (GvHD) by MSCs is Dependent on Inflammatory Cytokines and NOProduction

Mice were injected in the footpad with OVA alone or OVA and MSCs fromiNOS-deficient or wild-type mice. The mice were then challenged in thefootpad with OVA and the resultant DTH response measured by footpadswelling. The results of this analysis indicated that administration ofwild-type MSCs resulted in reduced inflammation in the DTH response. Insharp contrast, iNOS-deficient MSCs not only did not reduceinflammation, but also actually enhanced the DTH response in comparisonto challenged mice not injected with MSCs (FIG. 2 ). Histologicalanalysis of the footpads showed reduced indicators of inflammation inskin from animals co-injected with wild-type MSCs, while thoseco-injected with iNOS^(−/−) MSCs had increased fluid and leukocyteinfiltration at the site of inflammation. This experiment not onlydemonstrates the requirement for NO in the suppression of an immuneresponse, but also shows that, in the absence of NO production,MSC-mediated chemotaxis enhances inflammation, and could be used toboost local immune responses such as to promote the efficacy of vaccinesor provoke effective immune responses to tumors using inhibitors to iNOSand IDO.

One of the striking effects of immunosuppression by MSCs is the abilityto suppress graft-versus-host disease (GvHD) (Le Blanc, et al. (2004)supra; Le Blanc & Ringden (2006) supra). To investigate whethercytokine-induced NO production by MSC results in immunosuppression invivo, 5×10⁶ nucleated bone marrow cells and 5×10⁶ splenocytes fromC57BL/6 mice were injected into lethally irradiated F1 (C57BL/6×C3H)mice to established the mouse GvHD model. All recipient positive-controlmice developed extensive GvHD (wasting, ruffled hair, and hunched back)between days 15 and 22, while the negative controls that receivedsyngeneic F1 bone marrow were unaffected.

When F1 mice were treated with MSCs (0.5×10⁶ cells derived from donormice injected i.v. on days 3 and 7) after bone marrow transplantation,there was significant protection from GvHD; all MSC-treated micesurvived for at least 33 days and some for more than 75 days. Incontrast, F1 mice treated with MSCs derived from iNOS^(−/−) or IFNγR1^(−/−) mice were not protected, as their survival was not differentfrom untreated positive controls (FIG. 3 ). This lack of protection byMSCs deficient in IFNγ R1 or iNOS indicates that IFNγ and NO productionare essential for MSC-mediated immunosuppression in vivo.

Since in vitro results indicated that IFNγ acts together with either oneof the three cytokines, TNFα, IL-1α, or IL-1β to induce theimmunosuppressive function of MSCs, the role of these cytokines wasexamined in MSC-mediated protection from GvHD. Mice were injected withneutralizing antibodies against these cytokines or L-NMMA for 7 daysafter wild-type MSC infusion, and GvHD was allowed to develop. Bothanti-IFNγ and L-NMMA caused significant reversal of MSC-mediatedprotection from GvHD (FIG. 3 ), while negative control mice showed noadverse effect in response to these treatments.

The effect of a 3-antibody cocktail against TNFα, IL-1α, and IL-1β wasless dramatic, not reaching statistical significance (FIG. 4 ). Thisresult further implicates IFNγ and NO production, but is equivocal forthe other cytokines. It is important to recognize, however, that besidessynergizing with IFNγ to induce immunosuppression by MSCs, TNFα and IL-1are also important factors in the normal pathogenesis of GvHD. In fact,it has been reported that neutralization of either TNFα or IL-1 canlessen the severity of GvHD (Hattori, et al. (1998) Blood 91:4051-4055;McCarthy, et al. (1991) Blood 78:1915-1918). Therefore, it was somewhatexpected that protection from GvHD was not reversed to a greater extentby these antibodies.

Histological examination of the severity of inflammation in variousorgans from these mice was also examined 14 days after bone marrowtransplantation. The extent of observed leukocyte infiltrationcorrelated well with the survival results; GvHD-induced mice showedincreased numbers of lymphocytes in the liver, lungs, and skin, whilethey were nearly absent in those treated with MSCs. In addition,protection by MSC was almost completely reversed by anti-IFNγ andL-NMMA, while the 3-antibody cocktail against TNFα, IL-1α and IL-11 wereless effective. Together, the findings from these GvHD experiments, aswell as those from the DTH studies, clearly indicate a role for IFNγ andNO in MSC-mediated immunosuppression in vivo.

Example 7

Tumor-Derived MSC-Like Lymphoma Stromal Cells are Immunosuppressive

Since the tumor cells in lymphoma are not adherent, it is possible toisolate tumor stromal cells from lymphomas developed in p53+/− mice. Itwas observed that these cells can be passaged in vitro and can bedifferentiated into adipocytes and osteoblast-like cells. Interestingly,like bone marrow derived MSCs, these tumor stromal cells are alsoimmunosuppressive and can effectively inhibit the proliferation ofant-CD3-activated splenocytes. This immunosuppressive effect was alsodependent on IFNγ+TNF α and NO, since anti-IFNγ IFNγ and iNOS inhibitorscould reverse the immunosuppressive effect.

Example 8

Lymphoma Stromal Cells (LSCs) Promote Lymphoma Development in aNO-Dependent Manner

To examine the effect of lymphoma stromal cells on tumor growth, 355B-cell lymphoma cell line (C3H-gld/gld background, 0.5×10⁶ cells/mouse)was co-injected with gld/gld mice-derived lymphoma stromal cells (C3Hbackground, P5, 0.25×10⁶ cells/mouse). It was observed that co-injectionof stromal cells significantly enhanced the mortality. Interestingly,administration of 1400 W (NOS inhibitor, 0.1 mg/mouse on day 0, 2, 4, 8,12, 16, 20, 24, and 28) significantly reverted the effect (FIG. 4 ).Therefore, the tumor stromal cells could significantly promote tumorgrowth.

Example 9

Combination of NOS Inhibitor with IFNγ Promotes Mouse Melanoma Therapy

To test the role of tumor stromal cell-produced NO on tumorimmunotherapy, B16-F0 melanoma cells were injected into C57BL/6 mice onday 0 (0.5×10⁶ cells/mouse). IFNγ (250 ng/mouse) or 1400 W (NOSinhibitor, 0.1 mg/mouse) were administrated by i.p. injection on day 4,8, 12, 16, 20. Mice survival was recorded when mice were moribund. Itwas observed that the combined therapy dramatically promoted mousesurvival (FIG. 5 ). Thus, IFNγ has dual roles in tumor development; oneis to prevent tumor development by producing some angiostatic factors orblocking some angiogenesis factor production, the other is to induceimmunosuppression by tumor stromal or other environmental cells throughproducing factors like NO, IDO, or PGE2. Thus, inhibition of one or moreof NO, IDO or PGE2 can dramatically enhance cancer treatment. Therefore,when immunotherapies such as those based on cytokines, vaccination,antibodies, dendritic cells, or T cells, are used to treat cancer, thetumor stromal cells might be responsible for the inability of thesetreatment to completely eradicate tumors in most cases. The combinedused of inhibitors to iNOS and IDO with immunotherapies could provideeffective ways to eradicate tumors.

Example 10

IL-17A Synergizes with IFN γ and TNFα to Induce a High Expression of theImmunosuppressive Effector Molecule iNOS in Mouse Bone MarrowMesenchymal Stem Cells (BM-MSCs).

Mouse MSC-mediated immunosuppression is dependent on inflammatorycytokines. Without these cytokines, MSCs do not possess theimmunosuppressive effect, unless in the presence of inflammatorycytokines IFN γ with TNFα or IL-1 β, MSCs are stimulated to express theimmunosuppressive effector nitric oxide (NO), which is catalyzed byinducible NO synthase (iNOS), and several helper molecules-chemokinesand adhesion molecules. Chemokines and adhesion molecules retain T-cellsand other immune cells in the vicinity of MSCs, where high amount of NOsuppress the function of the immune cells.

Since the in vivo inflammatory environment contains various kinds ofinflammatory cytokines and growth factors in addition to the three typesof cytokines that we mentioned before, the in situ functions of MSCs inthe sites with tissue damage should be impacted by various cytokines inparticular microenvironment niches, the present inventors examined othercytokines especially those express at high levels in autoimmune diseasesand tissue injury.

Among several cytokines tested, IL-17A was found to greatly enhance iNOSexpression in the presence of IFNγ and TNFα. IL-17A is a criticalproinflamamtory cytokines found in many pathological conditions,however, little is known about how it influences the biology of MSCs. Asshown in FIG. 6 , at both mRNA and protein levels, IL-17A greatlyenhanced the expression of iNOS. This finding indicated that furthersupplement of IL-17A could be a potential strategy to enhance theimmunosuppressive activity of MSCs.

Example 11

IL-17A Enhances BM-MSC-Mediated Immunosuppression on T-CellProliferation.

To test if the IL-17A enhanced iNOS expression is functional or not, aMSC-T cell co-culture system was performed to evaluate theimmunosuppressive activity of MSCs. As shown in FIG. 7 , supplementationwith IFNγ and TNFα could decrease T-cell proliferation in a cytokineconcentration dependent manner. Strikingly, addition of IL-17A enhancedthe suppression of MSCs on T-cell proliferation. Therefore, IL-17A isfunctional in the enhancement of MSC-mediated immunosuppression.

Example 12

Materials and Methods

Reagents and Mice

Recombinant mouse IFNγ, TNFα, IL-17A, and antibodies against IL-17A werefrom eBiosciences (La Jolla, Calif.). Recombinant mouse IL-2 was fromR&D Systems (Minneapolis, Minn.). Antibodies against β-actin, GAPDH,iNOS, p-IκBα, p-P65, p-JNK, and p-ERK1/2 were from Cell SignalingTechnology (Danvers, Mass.). Antibody against Act1 was from Santa CruzBiotechnology (Dallas, Tex.). PMSF and actinomycin D were purchased fromSigma-Aldrich (St. Louis, Mo.).

C57BL/6 mice were maintained under specific pathogen-free conditions inthe vivarium with water and food provided ad libitum. All animalprotocols are approved by our Institutional Animal Care and UseCommittee.

Cells—MSCs were generated using the protocol as described in example 1.Briefly, tibia and femur bone marrow of 6-8 week old wild-type orauf1^(−/−) mice was harvested. Cells were cultured in DMEM mediumsupplemented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin, and 100μg/ml streptomycin (complete medium, all from Invitrogen, Carlsbad,Calif.). All nonadherent cells were removed after 24 hours (hr), andadherent cells were maintained. Medium was changed every 2-3 days. Toobtain MSC clones, cells at confluence were harvested and seeded into96-well plates by limited dilution. Individual clones were then pickedand expanded. MSCs were capable of differentiating into adipocytes andosteocytes under the respective differentiation conditions. Cells wereused before the 15th passage.

T cell blasts were generated from naive splenocytes isolated fromC57BL/6 mice, and cultured in RPMI-1640 medium supplemented with 10%heat-inactivated FBS, 2 mM glutamine, 100 U/ml penicillin, 100 μg/mlstreptomycin, and 50 μM β-ME. Splenocytes (1×10⁶ cells/ml) wereactivated with anti-CD3 and anti-CD28 for 48 hr, and harvested, withsupernatant filtered (0.1 μm) and frozen. The cells were then culturedwith IL-2 (200 U/ml) alone for 48 hr.

Proliferation assay—To assay T cell proliferation, 0.5 Ci of3H-thymidine was added to each well of 96-well plates 6 hr beforetermination of the cultures by freezing. Plates were then thawed, cellswere harvested, and incorporated 3H-Tdr was assessed with a WallacMicrobeta scintillation counter (Perkin-Elmer, Waltham, Mass.).

Messenger RNA decay assays—Messenger RNA decay assays were performedessentially. MSCs were incubated with cytokine combinations for 6 hr.Actinomycin D (Act.D) was added into the medium at a final concentrationof 5 μg/ml to stop transcription. At various time points after additionof Act.D, cells were harvested for extraction of total RNA.

Levels of iNOS, CXCL1, CCL2, CXCL10 and IL-6 mRNAs were measured at eachtime point by quantitative RT-PCR and normalized to levels of 3-actinmRNA. Percentages of mRNA remaining for each time point were plottedversus time after Act.D addition. First order decay constants, k, weredetermined by nonlinear regression. The associated mRNA half-lives,t1/2, were calculated with the equation t1/2=ln 2/k.

RNA isolation and gene expression assay—Total RNA was isolated with theRNAprep Pure Cell/Bacteria Kit. First-strand cDNA synthesis wasperformed with a cDNA synthesis kit. The levels of mRNAs were measuredby quantitative RT-PCR (7900 HT; Applied Biosystems, Foster City,Calif.) with SYBR Green Master Mix and normalized to the level ofβ-actin mRNA. Sequences of forward and reverse primer pairs are asfollows:

iNOS, forward 5′-CAGCTGGGCTGTACAAACCTT-3′ (SEQ ID NO:1) and reverse

5′-CATTGGAAGTGAAGCGTTTCG-3′ (SEQ ID NO:2);

β-actin: forward 5′-CCACGAGCGGTTCCGATG-3′ (SEQ ID NO:3) and reverse

5′-GCCACAGGATTCCATACCCA-3′ (SEQ ID NO:4);

IL-6: forward 5′-GAGGATACCACTCCCAACAGACC-3′ (SEQ ID NO:5) and reverse

5′-AAGTGCATCATCGTTGTTCATACA-3′; (SEQ ID NO:6)

CXCL1: forward 5′-CTGCACCCAAACCGAAGTC-3′ (SEQ ID NO:7) and reverse

5′-AGCTTCAGGGTCAAGGCAAG-3′; (SEQ ID NO:8)

CCL2: forward 5′-TCTCTCTTCCTCCACCACCATG-3′ (SEQ ID NO:9) and reverse

5′-GCGTTAACTGCATCTGGCTGA-3′; (SEQ ID NO:10)

CCL5: forward 5′-TTTCTACACCAGCAGCAAGTGC-3′ (SEQ ID NO:11) and reverse

5′-CCTTCGTGTGACAAACACGAC-3′; (SEQ ID NO:12)

CXCL9: forward 5′-AGTGTGGAGTTCGAGGAACCCT-3′ (SEQ ID NO:13) and reverse

5′-TGCAGGAGCATCGTGCATT-3′; (SEQ ID NO:14)

CXCL10: forward 5′-TAGCTCAGGCTCGTCAGTTCT-3′ (SEQ ID NO:15) and reverse

5′-GATGGTGGTTAAGTTCGTGCT-3′(SEQ ID NO:16).

Western blotting analysis-Cells were washed twice with ice-cold PBS,harvested and lysed in the RIPA buffer (Millipore, Temecular, Calif.)containing a cocktail of protease inhibitors (Roche, Natley, N.J.) andPMSF (Sigma) for 30 min on ice. Lysates were clarified by centrifugationat 16,000 g for 15 minutes. Protein concentration of the supernatant wasdetermined by the Bradford assay (Bio-Rad, Hercules, Calif.).

Protein samples were diluted in 5×SDS loading buffer (250 mM Tris-HCl,pH6.8, 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5%β-mercaptoethanol) and fractionated in a 10% SDS-polyacrylamide gel.Proteins were electroblotted onto a nitrocellulose membrane (WhatmanInc., Clifton, N.J.) and incubated for 1 hr in 5% nonfat dry milkdissolved in TBST (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.05% Tween 20)at room temperature. The blotting membranes were incubated with primaryantibodies overnight at 4° C., extensively washed in TBST, incubatedwith HRP-conjugated secondary antibody (Cell Signaling) for 1.5 hr atroom temperature, and washed again with TBST. The blotting membraneswere developed with chemiluminescent reagents (Millipore, Billerica,Mass.) according to the instructions provide by the manufacturer.

Immunofluorescence detection of IL-17A receptor—Cultured MSCs were firstwashed with PBS and fixed with ice-cold methanol at −20° C. for 10 min.After a 10 min incubation with 0.3% Triton X-100 in PBS, cells wereblocked with 5% BSA for 1 hr at room temperature and incubated withprimary antibody anti-IL-17RA (Santa Cruz) overnight at 4° C. Afterwashed by PBS, cells were incubated with Alexa Fluor 594 conjugated goatanti-rabbit secondary antibody and DAPI (Invitrogen) for 1 hr at roomtemperature. Cells were then washed with PBS before photographing.

ConA-induced liver injury in mice—C57BL/6 mice (8-10 week old) wereintravenously injected with ConA (Vector Labs, Burlingame, Calif.) inPBS at 15 mg/kg to induce liver injury. MSCs (5×105) derived fromwild-type mice or auf1^(−/−) mice were treated with or without IFNγ,TNFα in the presence or absence of IL-17A (10 ng/ml for each cytokine)for 12 hr, and then intravenously administrated into mice that have beentreated with ConA for 30 minutes. Mice were euthanized and serum andliver tissues were sampled after another 7.5 hr. Serum alanineaminotransferase (ALT) activity was determined by an ALT detection.Formalin-fixed liver histological sections were stained with hematoxilin& eosin (H&E).

Liver mononuclear cells iIsolation and flow cytometry analysis—Livermononuclear cells (MNCs) were purified by a 40%/70% gradient and stainedwith anti-CD3-PE, anti-CD4-PerCP/Cy5.5, and anti-CD8a-APC (eBiosciences)for 30 min at 4° C. in stainging buffer (PBS, 3% FCS). For detection ofsurface expression of IL-17RA in cloned MSCs, cells were stained withanti-IL-17RA-PE (eBiosciences) and analyzed by flow cytometry on a FACSCalibur flow cytometer (Becton Dickinson, San Jose, Calif.).

Statistical analysis—Nonlinear regression and statistical analyses wereperformed with PRISM v5 software (GraphPad Software, Inc.). Comparisonsbetween samples were performed with the unpaired t test. Differenceswith P<0.05 were considered significant. (*, P<0.05; **, P<0.01; ***,P<0.001).

Results—IL-17A Enhances the Immunosuppressive Effect of MSCs

The instant example provides that the immunosuppressive function of MSCsis not innate but induced by proinflammatory cytokines in aconcentration-dependent fashion. A combination of IFNγ and one of threeother inflammatory cytokines—TNFα, IL-1α, or IL-1β—is required to enablethe immunosuppressive effects of MSCs. IL-17A is a pleiotropicproinflammatory cytokine known for its critical roles in thepathogenesis of various inflammatory and autoimmune diseases.Interestingly, IL-17A is also known to synergize with certain cytokinesto promote gene expression programs required for inflammation.Therefore, the present inventors examined whether IL-17A could synergizewith suboptimal concentrations of IFNγ and TNFα to induce theimmunosuppressive property of MSCs.

MSCs were cultured with various combinations of recombinant cytokinesIFNγ, TNFα, and IL-17A at low concentrations (2 ng/ml each) for 12 hr;CD4′T cell blasts were added to the cultures at a 1:20 ratio (MSC:Tcells), together with IL-2, and T cell proliferation was assessed by³H-thymidine incorporation (it is noted that T cell blasts proliferatein the presence of IL-2, but they do not produce cytokines withoutfurther TCR activation). It was then concluded that any of the threecytokines alone cannot induce immunosuppression in MSCs.

MSCs were first treated with the indicated combinations of recombinantcytokines IFNγ, TNFα, IL-17A (2 ng/ml each) for 12 hr, then coculturedwith CD4⁺ T cell blasts at a 1:20 ratio (MSC: T cells), andproliferation was assessed by ³H-thymidine incorporation after anadditional 12 hr. Accordingly, T cell proliferation is suppressed in thepresence of IFNγ and TNFα, and this suppression can be markedly enhancedby IL-17A (FIG. 9 A), demonstrating a novel immunomodulatory function ofIL-17A, a potent proinflammatory cytokine.

The mRNA expression of IL-17 receptor family members in MSCs or Raw264.7 (Macrophages) were then examined by RT-PCR. NC: No RT. As such,IL-17A signals through IL-17RA and IL-17RC, expression of both receptorsin MSCs was confirmed by RT-PCR (FIG. 9B), in which a mouse macrophagecell line Raw 264.7 was used as a positive control; cell surfaceexpression of IL-17RA was also confirmed by indirect immunofluorescencemicroscopy and flow cytometry (FIG. 9C, upper and lower panels,respectively).

Since the concentrations of inflammatory cytokines vary at differentstages of an inflammatory response, the concentration dependence of IFNγand TNFα on IL-17A-enhanced immunosuppression was further assessed. MSCswere cultured with the indicated concentrations of IFNγ and TNFα,without or with 10 ng/ml IL-17A. MSCs were then co-cultured with CD4⁺ Tcell blasts or the A1.1 T cell hybridoma to assess the effects on T cellproliferation. IL-17A was able to enhance the immunosuppressive effectof MSCs on T cells at IFNγ and TNFα concentrations as low as 1-2 ng/mleach (FIG. 9D, 9E).

Even at higher concentrations of IFNγ and TNFα (i.e., 10-20 ng/ml),IL-17A still improved immunosuppression, though the effect was lesspronounced. Nonetheless, MSCs were treated with IFNγ and TNFα (2 ng/ml)with graded concentrations of IL-17A, for 12 hr, and then coculturedwith T cell hybridoma A1.1 cells at a ratio of 1:10 for 12 hr.Accordingly, the low concentrations of IFNγ and TNFα (2 ng/ml each), aslittle as 0.5 ng/ml IL-17A was sufficient to elicit a dramatic decreasein T cell proliferation (p<0.05; FIG. 9 F).

This observation provides MSCs can suppress proliferation of activatedprimary splenocytes depending on the inflammatory cytokines secreted byT cells. Activation of T cells lead to the production of many cytokines,including IL-17A. To confirm that IL-17A contributes to theimmunosuppressive effect of MSCs, antibody against IL-17A was used toneutralize it in a MSC-activated splenocyte co-culture system. Whileproliferation of activated splenocytes was markedly inhibited by MSCs,this inhibition was partially reversed upon addition of antibody againstIL-17A, i.e., proliferation increased in the presence of antibody (FIG.9G). The optimal effect of antibody occurred with a MSC:splenocyte ratioof 1:40. With a MSC:splenocyte ratio of 1:20, the reversal was lesspronounced, but still statistically significant (p<0.01). Takentogether, these results indicated that IL-17A can enhance theimmunosuppressive effects of MSCs, particularly when they are culturedin lower concentrations of IFNγ and TNFα.

IL-17A Synergizes with Inflammatory Cytokines to Induce the Expressionof Immune Modulatory Genes in MSCs

Nitric oxide (NO) and chemokines, acting in concert, are the keymolecules mediating the immunosuppressive effects of MSCs. Chemokine andiNOS genes in MSCs are induced by IFNγ and TNFα. However, IL-17Aenhanced immunosuppression by MSCs cultured with IFNγ and TNFα (FIG. 9D, 9 E). This study was then designed to show that IL-17A synergizeswith IFNγ and TNFα to induce the expression of iNOS and chemokines inMSCs. To test this hypothesis, a population of MSCs were cultured withvarious combinations of IFNγ, TNFα, and/or IL-17A, and the effects onexpression of selected immune modulatory genes were assessed.

Compared with MSCs cultured with single or double combinations ofcytokines, addition of IFNγ, TNFα, and IL-17A dramatically increasedexpression of iNOS, IL-6, and CXCL1 at the mRNA level (FIG. 10A);Western blot analysis confirmed an increase in iNOS protein levels aswell (FIG. 10 B). However, the expression of other chemokines such asCCL5, CCL2, CXCL9, CXCL10, which play pivotal roles in theimmunosuppressive effects of MSCs were all unaffected by addition ofIL-17A (FIG. 10 C).

To confirm that the effects on gene expression were due to IL-17A, MSCswere cultured with supernatant from anti-CD3 and anti-CD28-activatedsplenocytes in the presence or absence of neutralizing antibody againstIL-17A. Addition of anti-IL-17A to supernatants blocked induction ofiNOS, IL-6, and CXCL1 gene expression without affecting CCL2, CCL5,CXCL9, or CXCL10 (FIG. 10D, 10E). Blocking signal transduction of IL-17Ausing Act1 knockdown MSCs further verified such conclusion.

The recruitment of the adaptor protein Act1 to IL-17RA is linked toIL-17A dependent signaling. Since the phosphorylation of IκBα, ERK, p65,JNK were impaired in these Act1 knockdown MSCs (FIG. 11A), IL-17A couldnot upregulate IFNγ+TNFα induced iNOS expression in Act1 knockdown MSCs(FIG. 11B, 11C). Meanwhile, the enhancement of immunosuppression byIL-17A in MSCs was also not seen in absence of Act1 (FIG. 11D). Thus,the effects on gene expression were due to IL-17A. Together, these dataindicated that IL-17A can synergize with IFNγ and TNFα to induce theexpression of genes that contribute to the immunosuppressive function ofMSCs.

IL-17A Reverses the Suppression of Gene Expression Imposed byRNA-Binding Protein AUF1

Messenger RNAs encoding iNOS and many cytokines/chemokines are rapidlydegraded, which limits the abundance of both the mRNAs and proteins.Activation of signaling pathways, particularly during immune responses,stabilizes many of these mRNAs to increase their expression. Indeed, amajor mechanism by which IL-17A induces expression of many inflammatorymediator genes is by stabilizing their mRNAs. Numerous proteins bind tospecific RNA sequences, usually in the 3′-UTR, and target the mRNAs forrapid degradation. AU-rich elements (AREs) comprise one such family ofmRNA degradation sequences. There are numerous proteins that bind AREsto elicit controlled expression of the mRNAs harboring them. Theseproteins include AUF1, HuR, KSRP, TIA-1/TIAR, and TTP. Some of thetargets of these proteins include the ARE-mRNAs encoding iNOS, IL-6, andCXCL1. The ARE-binding protein AUF1 consists of four isoforms—p37, p40,p42, and p45—which bind and regulate degradation of iNOS and IL-6 mRNAs.It was thus hypothesized that AUF1 may act to limit the expression ofiNOS and cytokine/chemokine mRNAs and that IL-17A may block thisactivity of AUF1 thereby increasing gene expression.

As such, cytokine-induced gene expression was compared between MSCsderive from bone marrow of auf1^(−/−) mice and wild-type mice. Cellswere cultured as before with combinations of cytokines, with or withoutIL-17A. While levels of iNOS, IL-6, and CXCL1 mRNAs were normally verylow in wild-type MSCs, addition of IL-17A (together with IFNγ and TNFα)induced significant increases in these mRNAs (FIG. 12A; p<0.001). Bycontrast, levels of these three mRNAs were much higher in auf1^(−/−)MSCs cultured with IFNγ+TNFα, and addition of IL-17A had little effecton mRNA levels (i.e., IL-17A increased their abundance less thantwofold). Likewise, IFNγ and TNFα were sufficient to maximally induceiNOS protein in auf1^(−/−) MSCs without the need for IL-17A, in contrastto wild-type MSCs (FIG. 12B, compare lanes 7 and 8 with lane 5).

Given the effects of AUF1 and IL-17A on gene expression, it was possiblethat knockout of AUF1 alone would be sufficient to provide the degree ofmRNA stabilization, and increased gene expression, that would normallyrequire IL-17A. Wild-type and auf1^(−/−) MSCs were cultured withIFNγ+TNFα, with or without IL-17A. After 6 hr, Act.D was added to stoptranscription.

At various time points, RNA was isolated from cells and levels ofindividual mRNAs were determined to assess mRNA decay kinetics. Inwild-type MSCs, iNOS, IL-6, and CXCL1 mRNAs were relatively unstablewith half lives of 4.3±1.4 hr, 0.7±0.1 hr, and 0.59±0.08 hr,respectively; IL-17A led to a twofold stabilization of all three mRNAs(FIG. 13A; p<0.05 for each). The CCL2 and CXCL10 mRNAs, which did notrespond to IL-17A (see FIG. 13C), were not stabilized by IL-17A, aswould be expected (FIG. 13A; t1/2=˜2 hr for both mRNAs, with or withoutIL-17A).

In contrast to wild-type MSCs, knockout of AUF1 strongly stabilizediNOS, IL-6, and CXCL1 mRNAs (FIG. 13B; t1/2>10 hr for iNOS and IL-6;t1/2=4.3±0.6 hr for CXCL1). IL-17A had no effect on the half-lives ofiNOS and IL-6 mRNAs (FIG. 13B), while it stabilized CXCL1 mRNA at leasttwofold (FIG. 13B; t1/2>10 hr versus 4.3±0.6 hr without IL-17A; seeDiscussion). These mRNA decay data indicated that, (i) AUF1 normallypromotes degradation of iNOS, IL-6, and CXCL1 mRNAs in MSCs and IL-17Acauses their stabilization; (ii) stabilization of these mRNAs byknockout of AUF1 is comparable in magnitude to the stabilizing effectsof IL-17A on mRNAs in wild-type MSCs; and (iii) AUF1 knockout appears toobviate a requirement for IL-17A to induce iNOS/chemokine geneexpression. Thus, AUF1 may serve as a control point through which IL-17Amust act to elicit its effects on MSC gene expression, and possibly theultimate immunosuppression, which is examined next.

Effects of AUF1 on Immunosuppression by MSCs In Vitro

Given that AUF1 knockout induced chemokine gene expression without theneed for IL-17A (see FIGS. 12 and 13 ), it was hypothesized thatculturing auf1^(−/−) MSCs with IFNγ+TNFα alone would be sufficient tophenocopy the immunosuppressive activity of wild-type MSCs cultured withall three cytokines. To address this hypothesis, wild-type andauf1^(−/−) MSCs were cultured with IFNγ+TNFα, with or without IL-17A,and then co-cultured with the A1.1 T cell hybridoma for assays of T cellproliferation. IL-17A increased the immunosuppressive activity ofwild-type MSCs compared with cells cultured without it; however,IFNγ+TNFα was sufficient to induce maximal immunosuppressive activity ofauf1^(−/−) MSCs and IL-17A did not further enhance the immunosuppressiveeffect (FIG. 14 .A, 14B). These results, considered together, areconsistent with observations that AUF1 limits iNOS andcytokine/chemokine gene expression; IL-17A reverses this effect toenhance immunosuppression by MSCs.

IL-17A Enhances the Therapeutic Effect of MSCs in Mice Suffering fromConA-Induced Liver Injury in an AUF Dependent Manner

The inventors next examined the effects of IL-17A on immunosuppressionin vivo by wild-type and auf1^(−/−) MSCs. ConA-induced live injury inmice is a well-described in vivo model of autoimmune hepatitis mainlymediated by T cells. Since prior results showed that IL-17A candramatically enhance the immunosuppressive effect of MSCs in an in vitrosystems, it was expected that IL-17A could enable MSCs a bettertherapeutic effect in treating ConA-induced liver injury in mice.Accordingly, wild-type and auf1^(−/−) MSCs with and without IFNγ+TNFα,in the presence or absence of IL-17A, for 12 hr were first treated andthen intravenously injected into mice received ConA injection 30 minearlier. Compared with untreated or IFNγ+TNFα pretreated wild-type MSCs,IFNγ+TNFα and IL-17A pretreated wild-type MSCs could substantiallyameliorate liver damage with sharply reduced serum ALT activity andliver necrosis and inflammation (FIG. 15A, 15D). However, as forauf1^(−/−) MSCs, only IFNγ+TNFα pretreatment will elicit maximaltherapeutic effect in ConA induced liver injury, without the need forIL-17A (FIG. 15A, 15D). Consistent with the pattern of serum ALTactivity, mononuclear cells as well as CD3⁺CD4⁺ and CD3⁺CD8⁺ T cellsinfiltration in liver were also dramatically decreased in miceadministered by wild-type MSCs pretreated by IFNγ+TNFα with IL-17A, orauf1^(−/−) MSCs pretreated by IFNγ+TNFα with or without IL-17A (FIG.15B, 15C). Therefore, one of ordinary skill in the art can appreciate anew and novel therapy in treating ConA-induced liver damage, byutilizing MSCs pretreated by IFNγ+TNFα, together with IL-17A, and theeffect of IL-17A was exerted in an AUF1 dependent manner.

Example 13

IL-17A Promoted the iNOS Expression Through Enhancing the mRNA Stability

To investigate the mechanism of how IL-17A enhances iNOS expression andimmunosuppression by MSCs, the RNA stability of iNOS under cytokineinduction was studied. As shown in FIG. 16A, iNOS mRNA half decay timeis about 2.5 hours in the IFNγ+TNFα treatment group. Intriguingly,supplement with IL-17A completely protected iNOS mRNA within the timepoints tested.

In mammals, many mRNAs encoding inflammatory proteins could bedestabilized by AU-rich elements (AREs) present in their 3′-untranslatedregions. Rapid mRNA degradation occurs with the association of,ARE-binding proteins (AUBPs) with these mRNAs. AUFI, theARE/poly(U)-binding/degradation factor 1, is one of thebest-characterized AUBPs, which binds to many ARE-mRNAs to mediatedegradation. It was suspected that AUFI is critical for the observationsnoted in IL-17A-mediated iNOS overexpression in MSCs.

To test this, AUF1 was knocked down with siRNA in MSCs and treated withIFNγ+TNFα with and without IL-17A. In wild type MSCs IL-17A strikinglyinduced the iNOS expression, whereas the absence of AUF1 largelyabrogated this effect, indicating the importance of AUF1 inIL-17A-mediated iNOS expression in MSCs. Thus, IL-17A is capable ofstabilizing iNOS mRNA in MSCs, which provide a novel method toeffectively enhance the MSC-mediated therapy in clinical settings.

Type I Interferons and Fibroblast Growth Factor (FGF-2) Serve asNegative Regulators on MSC-Mediated Immunosuppression ThroughDown-Regulation of iNOS Expression.

As described above, IL-17A could be a potential factor to enhanceMSC-mediated immunosuppression. However, in many cases, suchimmunosuppressive effect in vitro and invivo, either positively ornegatively needs to be controlled. The available growth factors andcytokines were screened, and two factors were found strikinglydown-regulating MSC-mediated immunosuppression: type I interferons andfibroblast growth factor (FGF-2).

These two factors could potentially inhibit the immunosuppressive effectof MSCs towards T-cell proliferation (FIG. 16 ). Further analysisrevealed that, supplement of either of these cytokines was able tostrikingly reduce the expression of iNOS protein and NO production (FIG.17 ).

These findings substantiate methods to negatively control MSC-mediatedimmunosuppression. Accordingly, antibodies against type I interferonsand FGF can be used to boost the immunosuppressive effect of MSCs.

Construction of Human IDO-Expressing Mouse iNOS^(−/−) Cells(Humanized-IDO MSCs).

There is a species variation for MSC-mediated immunosuppression: NO isthe effector molecule for mouse MSCs, whereas human and primate MSCsutilize indoleamine 2,3-dioxygenase (IDO) as the suppressive effectormolecule.

Since mouse MSCs do not express indoleamine 2,3-dioxygenase (IDO) afterinflammatory cytokine stimulation, it is hard to study the biologicalrole of IDO in the mouse system. To circumvent this problem, mouseiNOS−/− MSCs were transfected with human IDO gene under the control ofmouse iNOS promoter. This allowed the expression of IDO in mouse MSCsupon inflammatory cytokine stimulation. Stable human IDO expressingmouse iNOS−/− MSCs have been successfully generated, with theverification of the high IDO expression under the stimulation by mouseinflammatory cytokines. With these humanized-IDO MSCs, IDO is shown tobe immunosuppressive in mouse MSCs in vitro and in vivo. Humanized-IDOmice were generated from iNOS−/− mice.

Human IDO gene is also being used to replace the mouse iNOS gene innormal mice in such the expressing of IDO is controlled by the mouseiNOS gene regulatory machineries, while the iNOS gene is silenced forfurther studying the pharmacology, cancer therapy, and assessing immuneresponse and immune related pathogenesis.

Example 14

Transducing a Population of MSCs to Release Functional IFN α

In this example, inventors transduced MSCs with lentivirus encoding GFP(MSC-GFP) or GFP together with mouse IFNα (MSC-IFNα). Over 90% cellswere successfully transduced, as shown by GFP expression on flowcytometry (FIG. 18A). No apparent changes in morphology andproliferation rate between MSC-GFP and MSC-IFN α were observed. Toexamine the IFN production level of MSC-IFNα, levels of IFNα wasquantified in the supernatant of MSCs cultured at 5×105 cells per ml for48 h (FIG. 18 B).

ELISA analysis showed that there was 19 ng/ml of IFNα in the supernatantof MSC-IFNα while no IFN α was detected in the supernatant of MSC-GFPcultured under the same condition. To test whether IFN α released byMSC-IFNα possesses biological function, the expression of MHC I moleculeH-2Kb on MSC surface was assessed by flow cytometry. IFN α increases theexpression level of H-2Kb.

FIG. 18 C elaborates that the expression of H-2Kb was low in MSC-GFP, anintrinsic property of MSCs. However, surprisingly after treatment withrecombinant IFN α, the expression of H-2Kb was dramatically increased inMSC-GFP. Similar increased expression of H-2b was also observed on cellstreated with the supernatant of MSC-IFNα. Correspondingly, H-2Kb surfaceexpression on MSC-IFNα was also increased to similar level as that ofMSC-GFP treated with IFNα. These data demonstrated that MSC-IFN αproduced biologically functional IFNα.

MSC-IFNα Exerted Potent Anti-Tumor Effect In Vivo.

To investigate the effect of MSC-IFN-α on tumor growth in vivo, mouseB16 melanoma model was employed. In this system, all cells and mice arein the C57BL/6 background. 1×10⁶ B16 melanoma cells were inoculatedalone, or with either 1×106 MSC-GFP or MSC-IFN-α intramuscularly, andtumors were removed and weighed twelve days later. It was unexpectedlyobserved that MSC-IFN α completely halted tumor growth, while MSC-GFPslightly enhanced tumor growth (FIG. 19A). To examine the potency of theMSC-IFNα, 1×10⁶ B16 melanoma cells with different numbers of MSC-IFNαwere injected to the animals. Surprisingly, even 1×10⁴ MSC-IFN α (at theratio of MSC-IFNα: B16=1:100) could still potently prevent tumor growthin vivo (FIG. 19B). Moreover, all mice inoculated with tumor cells alonedied within thirty days, while nearly half of mice received tumor cellstogether with MSC-IFNI survived for more than 100 days (FIG. 19C).

When MSC-IFNα cells were injected three or four days after B16 melanomacell inoculation, tumor growth was also effectively inhibited (FIGS. 19Dand 19E). To compare the anti-tumor capacity of MSC-IFNα withrecombinant IFNα, mice with 5 g recombinant IFNα (50,000 U) or 1×10⁶MSC-IFNα three days after B16 cell inoculation. Based on our in vitroassay, we roughly estimate that the 1×106 injected MSC-IFNα cells onlyproduce around 19 ng of IFNα daily. This is far below the 5 grecombinant IFNα injected. This observation is significant, as inventorsobserved that even with this low amount of IFN α produced (250 foldslower than the amount of recombinant IFNα injected), MSC-IFNα had muchmore potent anti-tumor effect than recombinant IFNα (FIG. 19F). RepeatedIFN α administration further exerted potent anti-tumor affect in vivo(Supplemental FIG. 18 ). These data clearly demonstrated thatIFNα-secreting MSCs possess highly potent anti-tumor activity in vivo.

MSCs Persisted in the Tumor, Decreased Tumor Cell Proliferation andInduced Tumor Cell Apoptosis.

To further investigate the mechanisms of the potent anti-tumor effect ofMSC-IFN.α, the fate of the administered MSC-IFN α in vivo was tracked.Accordingly, MSC-IFN α was labeled with luciferase, whose activity wasmonitored in vivo with live imaging technology sing Berthod NC100imaging system.

When co-injected with B16 cells, MSC-IFN α persisted only in tumors forover two weeks with gradual decrease (FIGS. 20A and 20B). Consideringthe potent anti-tumor effect of MSC-IFN α (still effective at the ratioof MSC-IFN α: B16=1:100), it is believed that SC-IFNα stay inside tumorsand consecutively secret low but effective concentration of IFNα locallyin the tumor for at least two weeks.

Those of ordinary skill in the art can appreciate the superiority ofthis affect to the short half-life and high dose requirement foradministration of recombinant IFNα in vivo. When tumors were examinedhistologically, massive lymphocyte infiltration was found in the B16plus MSC-IFNα Group.

MSC-IFNα inhibited tumor cell proliferation as shown by the decreasedratio of Ki-67-positive cells and increased tumor cell apoptosis, asshown by TUNEL assay (FIG. 20C).

The Anti-Tumor Activity of MSC-IFNα was Largely Immuno-Dependent.

The direct effect of recombinant IFN α on tumor growth in vitro wasstudied. It is found that recombinant IFNα only inhibited B16 melanomacells marginally even at high concentrations (up to 100 ng/ml, comparedto 19 ng/ml produced by MSC-IFNα) (FIG. 21A). Therefore, considering thecomplete tumor growth inhibition observed in vivo by MSC-IFNα, inventorsreasoned that there should be other mechanisms involved in addition tothe direct inhibition of tumor growth.

To test whether the immune system played any roles in the anti-tumoreffect of MSC-IFNα, B16 melanoma cells were inoculated alone, or witheither MSC-GFP or MSC-IFNα into wild-type and immunodeficient NOD-SCIDmice in parallel, and compared tumor growth in these mice. In wild-typemice, MSC-IFNα completely inhibited tumor growth (FIG. 21B), while inimmunodeficient mice the tumor inhibition effect of MSC-IFNα was greatlyabolished (FIG. 21C). To more clearly analyze the role of the immunesystem in the anti-tumor effect of MSC-IFN α, we injected less MSC-IFNαtogether with B16 tumor cells so as to minimize the contribution ofdirect tumor inhibition. When low numbers of MSC-IFN α (1/100 of tumorcells) were used, tumor growth was still effectively inhibited in wildtype mice (FIG. 21D); however this effect completely disappeared inimmunodeficient mice (FIG. 21E).

The inventors then tested whether NK cells were involved in theanti-tumor effect of MSC-IFNα by depleting NK cells with anti-asialo GM1antibody. Surprisingly, tumor growth was effectively inhibited incontrol mice; however, this inhibition was greatly reversed in micetreated with NK cells depletion antibody (FIG. 21F). CD8+ T cells alsocontributed to the anti-tumor effect of MSC-IFNα, as shown by thediminished inhibition of tumor growth by MSC-IFNα in CD8⁺ T cellsdeficient mice, β2m knockout mice (FIG. 21G). These data clearly showedthe immune system is critical in the anti-tumor effect of MSC-IFNα, inaddition to its direct effect on tumor cells.

In this study, IFNα was delivered into tumor via MSCs in normal mice. Insuch immune competent mice, IFNα was found to exert its effect throughpromoting anti-tumor immunity. Even low number of IFNα-secreting MSCshad the ability to inhibit one-hundred folds more tumor cell growth innormal mice, but not in immunodeficient mice. Furthermore, both NK cellsand CD8+ T cells were shown to play an important role in the anti-tumoreffect of IFNα-secreting MSCs in vivo.

IFNα could overcome the immunosuppression of MSCs. Accordingly, it iscontemplated that IFNα effectively reverses on the immunosuppressiveproperty of MSCs induced by IFNγ and TNFα. The long-term existence ofMSCs-IFNα in tumor avoided frequent injection as seen with IFNα. The lowbut effective level of IFNα released by MSCs-IFNα is unlikely to causeany side effects. Those of ordinary skill in the art can appreciate thatMSCs engineered to express immune stimulating factors hold great promisefor tumor therapy in the future.

While the invention has been described with references to specificembodiments, modifications and variations of the invention may beconstrued without departing from the scope of the invention, which isdefined in the following claims.

What is claimed is:
 1. A method for inducing an immune response in apatient in need thereof comprising administering to said patient aneffective amount of: (a) a population of isolated trained mesenchymalstem cells; (b) isolated IFNγ; and (c) an effective amount of at leastone cytokine for inducing an immune response, the at least one cytokineselected from the group consisting of interleukin-1 alpha (IL-1 α),interleukin-1 beta (IL-1 β), Type 1 interferon alpha (IFN-I α), Type 1interferon beta (IFN-I β), tumor necrosis factor alpha (TNF α),transforming growth factor beta (TGF β), and fibroblast growth factor(FGF).
 2. A method of inducing an immune response comprisingadministering to a subject in need thereof an effective amount of apopulation of isolated trained mesenchymal stem cells prepared by aprocess comprising the steps of: (i) obtaining multipotent progenitorcells; (ii) culturing said multipotent progenitor cells in a medium;(iii) separating mesenchymal stem cells from differentiated cells insaid medium; and (iv) activating at least a subset of said separatedmesenchymal stem cells by exposing it to isolated IFNγ and an effectiveamount of at least one cytokine for inducing an immune response, the atleast one cytokine selected from the group consisting of IL-1 α, IL-1 β,IFN-I α, IFN-I β, TNF α, TGF β, and FGF for a sufficient period of timesuch that the activated mesenchymal stem cells are immunomodulatory. 3.A method for inducing an immune response in a patient in need thereofcomprising administering to said patient a composition comprising aneffective amount of a population of isolated trained mesenchymal stemcells activated by exposure to IFNγ and an effective amount of at leastone cytokine for inducing an immune response, the at least one cytokineselected from the group consisting of IL-1 α, IL-1 β, TNF α, IFN-I α,IFN-I β, TGF β, and FGF for sufficient amount of time for a sufficientperiod of time such that the activated mesenchymal stem cells areimmunomodulatory.